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The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses. Editor's summary: CD45 is a cell surface phosphatase expressed by T cells and involved in regulating TCR signaling. CD45 does not have a known ligand but is targeted by various viruses, including the adenovirus E3/49K protein, as a strategy to evade immunity by suppressing T cell responses. Borowska et al. determined the cryo-EM structure of CD45 with the E3/49K protein and observed that the viral protein assembles as "beads on a string" that cross-link CD45 dimers, thus causing a conformational shift preventing intracellular phosphatase activity. These structural insights led to the engineering of CD45 surrogate ligands that modulate T cell activation by dimerizing CD45 in different orientations. —Christiana N. Fogg [ABSTRACT FROM AUTHOR]

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months.sup.1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. .sup.3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT.sup.5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies. CAR T cells administered intracerebroventricularly or intratumorally exhibit more rapid kinetics, reduced systemic toxicity and greater therapeutic potency as compared to intravenously delivered CAR T cells in atypical teratoid/rhabdoid tumor xenograft mouse models., Author(s): Johanna Theruvath [sup.1] , Elena Sotillo [sup.1] , Christopher W. Mount [sup.2] , Claus Moritz Graef [sup.1] , Alberto Delaidelli [sup.3] , Sabine Heitzeneder [sup.1] , Louai Labanieh [sup.1] [...]

Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies has surpassed expectations, driving an ever-expanding number of clinical trials and the first US Food and Drug Administration approvals of cell therapies for the treatment of cancer. This experience has illuminated some generalizable requirements for CAR T cell efficacy as well as the interplay between disease biology and clinical outcomes. Major CAR intrinsic variables affecting T cell behavior have been defined, and mechanisms of tumor resistance are increasingly understood. Here, we review the clinical experience with CAR T cells amassed to date, including but not limited to B cell malignancies, emphasizing factors associated with efficacy, resistance and major barriers to success. We also discuss how these insights are driving next-generation clinical trials, including those in solid tumors. Clinical application of CAR T cells to cancer therapy has met with much success, but future trials must build on current understanding to overcome therapy resistance, Author(s): Robbie G. Majzner [sup.1] [sup.2] , Crystal L. Mackall [sup.1] [sup.2] [sup.3] Author Affiliations: (1) Department of Pediatrics, Stanford, USA (2) Stanford Cancer Institute, Stanford, USA (3) Department of [...]

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M).sup.1-5 are aggressive and universally fatal pediatric brain cancers.sup.6. Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies.sup.7-10, and recent results suggest benefit in central nervous system malignancies.sup.11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain.sup.14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M.sup.+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2.sup.lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials.sup.15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M.sup.+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers. Lethal pediatric tumors bearing a particular histone H3 mutation upregulate the disialoganglioside GD2, thereby making these tumors susceptible to chimeric antigen receptor T cell-based immunotherapy., Author(s): Christopher W. Mount [sup.1] [sup.2] [sup.3] , Robbie G. Majzner [sup.4] , Shree Sundaresh [sup.1] , Evan P. Arnold [sup.1] , Meena Kadapakkam [sup.4] , Samuel Haile [sup.4] , [...]

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving [greater than equal ro]1 x 10[sup.6] CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19[sup.dim] or CD19[sup.-] B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22[sup.+] cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function., Author(s): Terry J Fry (corresponding author) [1]; Nirali N Shah [1]; Rimas J Orentas [1]; Maryalice Stetler-Stevenson [2]; Constance M Yuan [2]; Sneha Ramakrishna [1]; Pamela Wolters [1]; Staci Martin [...]