RESUMEN
Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Péptido Hidrolasas , Receptores de Antígenos de Linfocitos T , Linfocitos T/patologíaRESUMEN
Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.
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Antígeno CD47 , Inmunoterapia Adoptiva , Neoplasias , Linfocitos T , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Antígeno CD47/genética , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Línea Celular Tumoral , Inmunoterapia Adoptiva/métodos , Macrófagos/citología , Macrófagos/inmunología , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Microambiente Tumoral/inmunología , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Activación de MacrófagosRESUMEN
Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor with fatal outcome. Tumor-associated macrophages and microglia (TAMs) have been found to be major tumor-promoting immune cells in the tumor microenvironment. Hence, modulation and reeducation of tumor-associated macrophages and microglia in GBM is considered a promising antitumor strategy. Resident microglia and invading macrophages have been shown to have distinct origin and function. Whereas yolk sac-derived microglia reside in the brain, blood-derived monocytes invade the central nervous system only under pathological conditions like tumor formation. We recently showed that disruption of the SIRPα-CD47 signaling axis is efficacious against various brain tumors including GBM primarily by inducing tumor phagocytosis. However, most effects are attributed to macrophages recruited from the periphery but the role of the brain resident microglia is unknown. Here, we sought to utilize a model to distinguish resident microglia and peripheral macrophages within the GBM-TAM pool, using orthotopically xenografted, immunodeficient, and syngeneic mouse models with genetically color-coded macrophages (Ccr2RFP) and microglia (Cx3cr1GFP). We show that even in the absence of phagocytizing macrophages (Ccr2RFP/RFP), microglia are effector cells of tumor cell phagocytosis in response to anti-CD47 blockade. Additionally, macrophages and microglia show distinct morphological and transcriptional changes. Importantly, the transcriptional profile of microglia shows less of an inflammatory response which makes them a promising target for clinical applications.
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Neoplasias Encefálicas/inmunología , Antígeno CD47/inmunología , Glioblastoma/inmunología , Microglía/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Fagocitosis , Receptores Inmunológicos/inmunología , Transducción de Señal/inmunología , Animales , Neoplasias Encefálicas/patología , Antígeno CD47/genética , Glioblastoma/genética , Glioblastoma/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Microglía/patología , Monocitos/inmunología , Monocitos/patología , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Receptores Inmunológicos/genética , Transducción de Señal/genéticaRESUMEN
Purpose To develop a positron emission tomography (PET)/magnetic resonance (MR) imaging protocol for evaluation of the brain, heart, and joints of pediatric cancer survivors for chemotherapy-induced injuries in one session. Materials and Methods Three teams of experts in neuroimaging, cardiac imaging, and bone imaging were tasked to develop a 20-30-minute PET/MR imaging protocol for detection of chemotherapy-induced tissue injuries of the brain, heart, and bone. In an institutional review board-approved, HIPAA-compliant, prospective study from April to July 2016, 10 pediatric cancer survivors who completed chemotherapy underwent imaging of the brain, heart, and bone with a 3-T PET/MR imager. Cumulative chemotherapy doses and clinical symptoms were correlated with the severity of MR imaging abnormalities by using linear regression analyses. MR imaging measures of brain perfusion and metabolism were compared among eight patients who were treated with methotrexate and eight untreated age-matched control subjects by using Wilcoxon rank-sum tests. Results Combined brain, heart, and bone examinations were completed within 90 minutes. Eight of 10 cancer survivors had abnormal findings on brain, heart, and bone images, including six patients with and two patients without clinical symptoms. Cumulative chemotherapy doses correlated significantly with MR imaging measures of left ventricular ejection fraction and end-systolic volume, but not with the severity of brain or bone abnormalities. Methotrexate-treated cancer survivors had significantly lower cerebral blood flow and metabolic activity in key brain areas compared with control subjects. Conclusion The feasibility of a single examination for assessment of chemotherapy-induced injuries of the brain, heart, and joints was shown. Earlier detection of tissue injuries may enable initiation of timely interventions and help to preserve long-term health of pediatric cancer survivors. © RSNA, 2017 Online supplemental material is available for this article.
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Antineoplásicos/efectos adversos , Enfermedades Óseas/inducido químicamente , Encefalopatías/inducido químicamente , Cardiopatías/inducido químicamente , Imagen por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/estadística & datos numéricos , Tomografía de Emisión de Positrones/métodos , Adolescente , Enfermedades Óseas/diagnóstico por imagen , Encefalopatías/diagnóstico por imagen , Supervivientes de Cáncer , Femenino , Cardiopatías/diagnóstico por imagen , Humanos , Masculino , Imagen Multimodal , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Factores de Riesgo , Sensibilidad y Especificidad , Integración de Sistemas , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Considering the improved outcome, a better understanding of the late effects in Wilms tumor survivors (WT-S) is needed. This study was aimed at evaluating renal function and determining the prevalence of clinical and subclinical renal dysfunction in a cohort of WT-S using a multimodal diagnostic approach. METHODS: Thirty-seven WT-S were included in this prospective cross-sectional single center study. To evaluate renal function, glomerular filtration rate (GFR) and urinary protein excretion were assessed. Additionally, kidney sonomorphology and blood pressure were analyzed. RESULTS: All examined WT-S (mean age 28.7 years, mean follow-up 24.8 years) had been treated with a combination of surgery and chemotherapy; 59.5% had received adjuvant radiotherapy. Impaired glomerular renal function was detected in a considerable proportion of WT-S, with age-adjusted cystatin-based GFR estimation below age norm in 55.9%. A lower cystatin-based estimated GFR (eGFR) correlated with longer follow-up time and higher irradiation dose. In 5 patients (13.5%) albuminuria was identified. Analysis of sonomorphology detected compensatory contralateral renal hypertrophy in 83.3% of WT-S. Chronic kidney disease (CKD) ≥ stage II was present in 55.9% of WT-S. Blood pressure measurements revealed arterial hypertension in 15 (40.5%) WT-S (newly diagnosed n=10). In 24.3% both CKD ≥ stage II and arterial hypertension were determined. CONCLUSION: Even though WT-S are believed to carry a low risk for end-stage renal disease, in this study, a remarkable number of WT-S presented with previously unidentified subclinical signs of renal function impairment and secondary morbidity. Therefore, it is important to continue regular follow-up, especially after transition into adulthood.
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Supervivientes de Cáncer/estadística & datos numéricos , Neoplasias Renales/fisiopatología , Riñón/fisiopatología , Insuficiencia Renal Crónica/epidemiología , Tumor de Wilms/fisiopatología , Adolescente , Adulto , Antineoplásicos/efectos adversos , Quimioterapia Adyuvante , Estudios Transversales , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Humanos , Riñón/diagnóstico por imagen , Riñón/efectos de los fármacos , Riñón/cirugía , Neoplasias Renales/mortalidad , Neoplasias Renales/terapia , Masculino , Persona de Mediana Edad , Nefrectomía/efectos adversos , Nefrectomía/métodos , Prevalencia , Estudios Prospectivos , Proteinuria/epidemiología , Proteinuria/fisiopatología , Proteinuria/orina , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/orina , Ultrasonografía , Tumor de Wilms/mortalidad , Tumor de Wilms/terapia , Adulto JovenRESUMEN
PURPOSE: Cerebrospinal cavernous malformations (CCMs) are vascular lesions characterized by dilated and leaky capillary caverns. CCMs can cause seizures, focal neurological deficits or acute intracranial hemorrhage; however, most patients are asymptomatic. CCMs occur either sporadically or as a familial autosomal-dominant disorder. We present a clinical and molecular study of a patient with distinctive cerebral and spinal cavernous malformations following radiochemotherapy for a malignant brain tumor. METHODS: The patient had multiple magnet resonance imaging (MRI) examinations of his brain and spine following radiochemotherapy for a primary intracranial germ cell tumor (GCT), as part of his oncologic follow-up. The MRI sequences included susceptibility-weighted imaging (SWI). The coding exons and their flanking intronic regions of KRIT1/CCM1 gene were analyzed for mutations by polymerase chain reaction (PCR) and direct sequencing. RESULTS: MRI revealed numerous cerebral and spinal microhemorrhages and pronounced cavernous malformations that progressed with subsequent follow-up imaging. Genetic analysis demonstrated a novel heterozygous KRIT1/CCM1 two base pair deletion (c.1535_1536delTG) in exon 14. This deletion leads to a frameshift with a premature stop codon at nucleotide position 1553 and a highly likely loss of function of the KRIT1 protein. CONCLUSION: We describe a patient with a novel heterozygous germ line loss of function mutation in KRIT1, which is associated with rapid-onset and highly progressive CCMs after radiochemotherapy for a malignant brain tumor.
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Quimioradioterapia/efectos adversos , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteína KRIT1/genética , Mutación con Pérdida de Función/genética , Análisis Mutacional de ADN , Estudios de Seguimiento , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico por imagen , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/terapia , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/terapia , Adulto JovenRESUMEN
Although neuro- and nephroblastoma are common solid tumors in children, the simultaneous occurrence is very rare and is often associated with syndromes. Here, we present a unique case of synchronous occurrence of neuro- and nephroblastoma in an infant with no signs of congenital anomalies or a syndrome. We performed genetic testing for possible candidate genes as underlying mutation using the next-generation sequencing (NGS) approach to target 94 genes and 284 single-nucleotide polymorphisms (SNPs) involved in cancer. We uncovered a novel heterozygous germline missense mutation p.F58L (c.172TâC) in the anaplastic lymphoma kinase (ALK) gene and one novel heterozygous rearrangement Q418Hfs(*)11 (c.1254_1264delins TTACTTAGTACAAGAACTG) in the Fanconi anemia gene FANCD2 leading to a truncated protein. Besides, several SNPs associated with the occurrence of neuroblastoma and/or nephroblastoma or multiple primary tumors were identified. The next-generation sequencing approach might in the future be useful not only in understanding tumor etiology but also in recognizing new genetic markers and targets for future personalized therapy.
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Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Renales/genética , Neoplasias Primarias Múltiples/genética , Neuroblastoma/genética , Tumor de Wilms/genética , Quinasa de Linfoma Anaplásico , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Humanos , Lactante , Neoplasias Renales/terapia , Masculino , Neoplasias Primarias Múltiples/terapia , Neuroblastoma/terapia , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Quinasas Receptoras/genética , Tumor de Wilms/terapiaRESUMEN
BACKGROUND: While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway. METHODS: We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models. RESULTS: CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment ('eat-me') signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION: Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.
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Neoplasias Cerebelosas , Glioblastoma , Meduloblastoma , Humanos , Ratones , Animales , Meduloblastoma/tratamiento farmacológico , FN-kappa B/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Proteína Glutamina Gamma Glutamiltransferasa 2 , Calidad de Vida , Fagocitosis , Macrófagos , Inflamación/metabolismoRESUMEN
OBJECTIVE: To assess diffusion and perfusion changes of the auditory pathway in pediatric medulloblastoma patients exposed to ototoxic therapies. STUDY DESIGN: Retrospective cohort study. SETTING: A single academic tertiary children's hospital. PATIENTS: Twenty pediatric medulloblastoma patients (13 men; mean age 12.0â±â4.8âyr) treated with platinum-based chemotherapy with or without radiation and 18 age-and-sex matched controls were included. Ototoxicity scores were determined using Chang Ototoxicity Grading Scale. INTERVENTIONS: Three Tesla magnetic resonance was used for diffusion tensor and arterial spin labeling perfusion imaging. MAIN OUTCOME MEASURES: Quantitative diffusion tensor metrics were extracted from the Heschl's gyrus, auditory radiation, and inferior colliculus. Arterial spin labeling perfusion of the Heschl's gyrus was also examined. RESULTS: Nine patients had clinically significant hearing loss, or Chang grades more than or equal to 2a; 11 patients had mild/no hearing loss, or Chang grades less than 2a. The clinically significant hearing loss group showed reduced mean diffusivity in the Heschl's gyrus (pâ=â0.018) and auditory radiation (pâ=â0.037), and decreased perfusion in the Heschl's gyrus (pâ=â0.001). Mild/no hearing loss group showed reduced mean diffusivity (pâ=â0.036) in Heschl's gyrus only, with a decrease in perfusion (pâ=â0.008). There were no differences between groups in the inferior colliculus. There was no difference in fractional anisotropy between patients exposed to ototoxic therapies and controls. CONCLUSIONS: Patients exposed to ototoxic therapies demonstrated microstructural and physiological alteration of the auditory pathway. The present study shows proof-of-concept use of diffusion tensor imaging to gauge ototoxicity along the auditory pathway. Future larger cohort studies are needed to assess significance of changes in diffusion tensor imaging longitudinally, and the relationship between these changes and hearing loss severity and longitudinal changes of the developing auditory white matter.
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Corteza Auditiva , Neoplasias Cerebelosas , Meduloblastoma , Ototoxicidad , Adolescente , Vías Auditivas/diagnóstico por imagen , Neoplasias Cerebelosas/diagnóstico por imagen , Neoplasias Cerebelosas/tratamiento farmacológico , Niño , Imagen de Difusión Tensora , Humanos , Imagen por Resonancia Magnética , Masculino , Meduloblastoma/diagnóstico por imagen , Meduloblastoma/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
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.
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Neoplasias Óseas , Antígeno CD47 , Animales , Línea Celular Tumoral , Humanos , Inmunoterapia , Ratones , Recurrencia Local de Neoplasia , Fagocitosis , Microambiente TumoralRESUMEN
Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (â¼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.
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Glipicanos/inmunología , Inmunoterapia Adoptiva , Neuroblastoma/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Glipicanos/metabolismo , Humanos , Inmunoterapia/métodos , Neuroblastoma/patología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
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.
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Receptores Quiméricos de Antígenos/metabolismo , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
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 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 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 ATRT5,6, 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.
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Antígenos B7/inmunología , Neoplasias Encefálicas/terapia , Vacunas contra el Cáncer/administración & dosificación , Inmunoterapia Adoptiva/métodos , Tumor Rabdoide/terapia , Teratoma/terapia , Adulto , Animales , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/patología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Células Cultivadas , Preescolar , Femenino , Feto/patología , Humanos , Lactante , Inyecciones Intraventriculares , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores Quiméricos de Antígenos/administración & dosificación , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Tumor Rabdoide/inmunología , Tumor Rabdoide/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Teratoma/inmunología , Teratoma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The long-term survival of osteosarcoma patients with metastatic or recurrent disease remains dismal, and new therapeutic options are urgently needed. The purpose of our study was to compare the efficacy of CD47 mAb plus doxorubicin combination therapy in mouse models of osteosarcoma with CD47 mAb and doxorubicin monotherapy. Forty-eight NOD scid gamma (NSG) mice with intratibial MNNG/HOS tumors received CD47 mAb, doxorubicin, combination therapy, or control IgG treatment. Twenty-four mice (n = 6 per group) underwent pre- and post-treatment magnetic resonance imaging (MRI) scans with the macrophage marker ferumoxytol, bioluminescence imaging, and histological analysis. Tumor ferumoxytol enhancement, tumor flux, and tumor-associated macrophages (TAM) density were compared between different groups using a one-way ANOVA. Twenty-four additional NSG mice underwent survival analyses with Kaplan-Meier curves and a log-rank (Mantel-Cox) test. Intratibial osteosarcomas demonstrated significantly stronger ferumoxytol enhancement and significantly increased TAM quantities after CD47 mAb plus doxorubicin combination therapy compared to CD47 mAb (P = 0.02) and doxorubicin monotherapy (P = 0.001). Tumor-bearing mice treated with CD47 mAb plus doxorubicin combination therapy demonstrated significantly reduced tumor size and prolonged survival compared to control groups that received CD47 mAb (P = 0.03), doxorubicin monotherapy (P = 0.01), and control IgG (P = 0.001). In conclusion, CD47 mAb plus doxorubicin therapy demonstrates an additive therapeutic effect in mouse models of osteosarcomas, which can be monitored with an immediately clinically applicable MRI technique.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Antígeno CD47/inmunología , Doxorrubicina/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Animales , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/inmunología , Línea Celular Tumoral , Óxido Ferrosoférrico/análisis , Humanos , Luminiscencia , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Imagen por Resonancia Magnética , Ratones Endogámicos NOD , Imagen Óptica , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/inmunología , Fagocitosis/efectos de los fármacosRESUMEN
CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivo are currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used "off label" as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p < 0.01) and significantly increased F4/80+CD80+ M1 macrophages on histopathology (p < 0.01). CD47 mAb-treated F4/80+ macrophages demonstrated significantly augmented phagocytosis of ferumoxytol nanoparticles (p < 0.01). Thus, we conclude that ferumoxytol-MRI can detect TAM response to CD47 mAb in mouse models of osteosarcoma. The ferumoxytol-MRI imaging test could be immediately applied to monitor CD47 mAb therapies in clinical trials.
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Anticuerpos Monoclonales/metabolismo , Antígeno CD47/genética , Inmunoterapia/métodos , Macrófagos/metabolismo , Imagen por Resonancia Magnética/métodos , Nanopartículas/metabolismo , Osteosarcoma/genética , Animales , Humanos , Ratones , Osteosarcoma/patologíaRESUMEN
PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. RESULTS: B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.
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Antígenos de Neoplasias/inmunología , Antígenos B7/inmunología , Neoplasias Encefálicas/etiología , Neoplasias Encefálicas/metabolismo , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos B7/antagonistas & inhibidores , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Inmunoterapia Adoptiva/métodos , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.
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Quinasa de la Caseína II/metabolismo , Neoplasias Cerebelosas/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteómica/métodos , Transducción de Señal , Anilidas/farmacología , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Línea Celular Tumoral , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Humanos , Estimación de Kaplan-Meier , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células 3T3 NIH , Naftiridinas/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Fenazinas , Fosfoproteínas/genética , Piridinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway.
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Proliferación Celular/genética , Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica , Meduloblastoma/genética , Receptor Notch1/genética , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Línea Celular Tumoral , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/metabolismo , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Receptor Notch1/inmunología , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The original version of this Article omitted Suzana A. Kahn, Siddhartha S. Mitra & Samuel H. Cheshier as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches.We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO, PTCH1 and SUFU, three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC50 of 1.3 µM.In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.