IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity.

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In Vivo Persistence and Antitumor Activity against Neuroblastoma.

PURPOSE: Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs. EXPERIMENTAL DESIGN: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma. RESULTS: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis. CONCLUSIONS: Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

Análisis de la calidad del registro de paro cardíaco en casos de responsabilidad médico legal en médicos generales, 1999-2007 / Quality of cardiac arrest records in cases of medical responsibility, 1999-2007

Antecedentes. El registro del paro cardíaco es una actividad que se debe acompañar en la reanimación cerebro cardio pulmonar. En Colombia no existe un formato de registro oficial de paro cardiorrespiratorio, lo que dificulta la investigación en reanimación y la conducción de los procesos de responsabilidad médica que se deriven de esta atención. Objetivo. Analizar la calidad del registro de paro cardíaco en casos de responsabilidad médico legal iniciada a médicos generales que tuvieron asesoría científica del Fondo para Auxilio de Demandas de la Sociedad Colombiana de Anestesiología y Reanimación (SCARE-FEPASDE), 1999-2007.Material y métodos. Se realizó una investigación tipo serie de casos, en donde se incluyeron los casos de paro cardiorrespiratorio que tuvieron demanda médico legal y que fueron atendidos por médicos generales, entre 1999 y 2007, y que requirieron asesoría jurídica por SCARE-FEPASDE. La información se registró en tablas de Excel®, según las recomendaciones Utstein. La calificación de calidad se hizo por tres médicos expertos en responsabilidad médica de manera independiente. Se utilizó un formato de calificación sobre la proporción de registros realizados sobre 25 ítem posibles. La calidad se consideró alta (mayor de 95 por ciento ítem), media (70-94 por ciento ítem) y baja (menor de 70 por ciento). En los casos de desacuerdo se realizó consenso. Resultados. Se incluyeron en total 83 casos. La calidad del registro del paro fue media en 49 casos (59 por ciento), baja en 32 casos (39 por ciento) y alta en dos casos.Conclusión. El registro de paro cardiorrespiratorio en casos de responsabilidad médica es de mediana calidad, por lo que no se alcanza a cumplir con los estándares internacionales y las guías al estilo Utstein.

Analgesia preventiva en mujeres programadaspara esterilización definitiva conelectrofulguración de trompas uterinas porlaparoscopia: Experimento clínico controlado, aleatorizado, con doble enmascaramiento / Preventive analgesia in women scheduled for definitive sterilization with electrofulguration of uterine tubes by laparoscopy Controlled, randomized clinical trial with double masking

Antecedentes: La analgesia preventiva continúa siendo fuente de discusión y desacuerdo entre los especialistas que manejan el dolor. Hay resultados contradictorios, incluidos algunos metaanálisis, sobre la efectividad de la analgesia preventiva con ketamina. Objetivo: Evaluar la eficacia de la ketamina preventiva en la cirugía ambulatoria de mujeres programadas para esterilización definitiva con electrofulguración por laparoscopia, en la disminución del dolor postoperatorio, comparada contra ketamina administrada de manera no preventiva. materiales y métodos: Entre diciembre de 2007 y enero de 2008 se incluyeron 130 mujeres de la Clínica Piloto de Profamilia, en Bogotá (Colombia), que deseaban planificar mediante ligadura de trompas. Fueron asignadas de manera aleatoria simple a dos grupos: a 71 pacientes se les administró ketamina antes de la incisión quirúrgica (0.25 mg/kg) y a 59 pacientes se les administró ketamina a la misma dosis al fnal del procedimiento. La administración de ketamina y la evaluación de los desenlaces se realizaron de manera enmascarada. Resultados: El nivel de dolor medido mediante la Escala Visual Análoga (EVA) fue similar para ambos grupos, aunque hubo una mayor proporción de pacientes con dolor al minuto 15 en el grupo de ketamina preventiva (19.7% vs. 6.8%, p=0.03). El grupo de ketamina no preventiva presentó más náusea (15% vs 10%) y mareo (46% vs. 37%), aunque esto no fue significativo (p>0,05) Conclusión: La eficacia de la ketamina preventiva para el alivio del dolor postoperatorio no fue diferenre a la de la ketamina no preventiva .

Background: The pre-emptive analgesia keeps being a source of discussion and discord between the spe-cialists in the pain management. There are contradictory results, even in meta-analysis, about effectiveness of preemptive analgesia with ketamine. Objective: To evaluate effectiveness of ketamine as preemptive analgesia in women under laparoscopic electrofulguration of Falopio tubes, in control of postoperative pain, compared with ketamine administered in a non preemptive way. material and methods: 130 women who wanted a defnitive contraceptive method as laparoscopic tubal ligation were included with the diagnosis of satisfed parity, from the Clínica Piloto de Profamilia, in Bogotá (Colombia), between december 2007 and january o2008. They were randomly assigned in two groups: 71 received preemptive ketamine (before initiating the surgical procedure, dose of 0,25 mg/kg) and the other 59 patients received ketamine, the same doses, at the end of the procedure. Both, administration of ketamine and evaluation of patients outcomes, were made in blinded form. Results: Patients were similar at the start of the study. The level of pain measured with a visual analog scale (VAS) was similar for both groups, there was a higher proportion of patients with pain in the preemp-tive ketamine group to the minute 15 (29.7% vs 6.8%) Ketamine non preemptive group, felt more nauseous (25% vs. 10% and seasickness (46% vs 37%), this was not signifcative (p>0.05)). Conclusions: The preemptive ketamine wasn't different in effectiveness to ketamine administered in non preemptive way for the relief of postoperative pain .