RESUMEN
Somatic variants in DNA damage response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM deficiencies underlie ataxia-telangiectasia (A-T), a disease manifested by radiosensitivity, immunodeficiency, and predisposition to lymphoid malignancies. Patients with A-T diagnosed with malignancies have poor tolerance to chemotherapy or radiation. In this study, we investigated chimeric antigen receptor (CAR) T cells using primary T cells from patients with A-T (ATM-/-), heterozygote donors (ATM+/-), and healthy donors. ATM-/- T cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM-/- CAR T-cells was observed. Retroviral transduction of the CAR in ATM-/- T cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR T-cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity. Significance: CAR T-cells are clinically approved genetically modified cells, but the control of genome integrity remains largely uncharacterized. This study demonstrates that ATM deficiency marginally impairs CAR T-cell function and results in high rates of chromosomal aberrations after retroviral transduction, which may be of concern in patients with DNA repair deficiencies.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Receptores Quiméricos de Antígenos , Retroviridae , Linfocitos T , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Humanos , Linfocitos T/inmunología , Retroviridae/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/inmunología , Transducción Genética , Daño del ADN , Inmunoterapia Adoptiva/métodosRESUMEN
CD19 CAR-T cells have led to durable remissions in patients with refractory B-cell malignancies; nevertheless, most patients eventually relapse in the long term. Many interventions aimed at improving current products have been reported, with a subset of them focusing on a direct or indirect link to the metabolic state of the CAR-T cells. We assessed clinical products from an ongoing clinical trial utilizing CD19-28z CAR-T cells from patients with acute lymphoblastic leukemia. CAR-T clinical products leading to a complete response had significantly higher mitochondrial function (by oxygen consumption rate) irrespective of mitochondrial content. Next, we replaced the carbon source of the media from glucose to galactose to impact cellular metabolism. Galactose-containing media increased mitochondrial activity in CAR-T cells, and improved in in-vitro efficacy, without any consistent phenotypic change in memory profile. Finally, CAR-T cells produced in galactose-based glucose-free media resulted in increased mitochondrial activity. Using an in-vivo model of Nalm6 injected mice, galactose-primed CAR-T cells significantly improved leukemia-free survival compared to standard glucose-cultured CAR-T cells. Our results prove the significance of mitochondrial metabolism on CAR-T cell efficacy and suggest a translational pathway to improve clinical products.
Asunto(s)
Galactosa , Inmunoterapia Adoptiva , Mitocondrias , Galactosa/metabolismo , Animales , Ratones , Mitocondrias/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Antígenos CD19/metabolismo , Linfocitos T/metabolismo , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD19 CAR-T cells have led to durable remissions in patients with refractory B-cell malignancies; nevertheless, most patients eventually relapse in the long term. Many interventions aimed at improving current products have been reported, with a subset of them focusing on a direct or indirect link to the metabolic state of the CAR-T cells. We assessed clinical products from an ongoing clinical trial utilizing CD19-28z CAR-T cells from patients with acute lymphoblastic leukemia. CAR-T clinical products leading to a complete response had significantly higher mitochondrial function (by oxygen consumption rate) irrespective of mitochondrial content. Next, we replaced the carbon source of the media from glucose to galactose to impact cellular metabolism. Galactose-containing media increased mitochondrial activity in CAR-T cells, and improved in vitro efficacy, without any consistent phenotypic change in memory profile. Finally, CAR-T cells produced in galactose-based glucose-free media resulted in increased mitochondrial activity. Using an in vivo model of Nalm6 injected mice, galactose-primed CAR-T cells significantly improved leukemia-free survival compared to standard glucose-cultured CAR-T cells. Our results prove the significance of mitochondrial metabolism on CAR-T cell efficacy and suggest a translational pathway to improve clinical products.
RESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. METHODS: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. RESULTS: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). CONCLUSIONS: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Antígenos CD19 , Tratamiento Basado en Trasplante de Células y Tejidos , Epigénesis Genética , Humanos , Inmunoterapia Adoptiva/efectos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.
RESUMEN
Autologous T cells engineered to express a chimeric antigen receptor (CAR) against the CD19 antigen are in the frontline of contemporary hemato-oncology therapies, leading to high remission rates in B-cell malignancies. Although effective, major obstacles involve the complex and costly individualized manufacturing process, and CD19 target antigen loss or modulation leading to resistant and relapse following CAR therapy. A potential solution for these limitations is the use of donor-derived γδT cells as a CAR backbone. γδT cells lack allogenecity and are safely used in haploidentical transplants. Moreover, γδT cells are known to mediate natural anti-tumor responses. Here, we describe a 14-day production process initiated from peripheral-blood mononuclear cells, leading to a median 185-fold expansion of γδ T cells with high purity (>98% CD3+ and >99% γδTCR+). CAR transduction efficacy of γδ T cells was equally high when compared to standard CAR-T cells (60.5 ± 13.2 and 65.3 ± 18.3%, respectively). CD19-directed γδCAR-T cells were effective against CD19+ cell lines in vitro and in vivo, showing cytokine production, direct target killing, and clearance of bone marrow leukemic cells in an NSG model. Multiple injections of γδCAR-T cells and priming of mice with zoledronate lead to enhanced tumor reduction in vivo. Unlike standard CD19 CAR-T cells, γδCAR-T cells were able to target CD19 antigen negative leukemia cells, an effect that was enhanced after priming the cells with zoledronate. In conclusion, γδCAR-T cell production is feasible and leads to highly pure and efficient effector cells. γδCAR-T cell may provide a promising platform in the allogeneic setting, and may target leukemic cells also after antigen loss.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Inmunoterapia Adoptiva/métodos , Leucemia , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Ingeniería Genética/métodos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción Genética/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Autologous T cells transduced with CD19-directed chimeric antigen receptors have recently been approved by several regulatory agencies for the treatment of relapsed and refractory leukemia and lymphoma, after demonstrating remarkable remission rate in advanced patients. The most common adverse events reported are cytokine-release syndrome (CRS), neurotoxicity, and hematologic toxicity. Here, we focus on early and late cytopenia occurring after CD19 CAR-T cells in 38 patients treated with CD19 CAR-T cells. Neutropenia, thrombocytopenia, and anemia occur frequently (94, 80, and 51%, respectively) after CAR-T cell infusion, and are associated with a biphasic nature, as in 93% of patients hematologic toxicity occurs after 21 days from cell infusion. Late hematologic toxicity was more common in patients with high grade CRS and in patients treated after a recent stem cell transplantation. Interestingly, since these events occur late after the lymphodepleting chemotherapy and after resolution of CRS, we found perturbations in SDF-1 levels to correlate with events of late neutropenia, likely associated with B-cell recovery.
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Antígenos CD19/efectos adversos , Enfermedades Hematológicas/inducido químicamente , Receptores Quiméricos de Antígenos/inmunología , Adolescente , Adulto , Antígenos CD19/inmunología , Niño , Preescolar , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Autologous CD19 chimeric-antigen receptor (CAR) T cells demonstrated remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (R/R ALL). Here, we report results from a phase 1b/2 study of in-house produced CD19 CAR with a CD28 costimulatory domain. Twenty-one patients with R/R ALL were enrolled, and 20 infused. The median age was 11 years (range, 5-48). Patients had a median of 4 prior regimens, including blinatumomab in 6 and prior stem-cell transplantation in 10. In total 8 patients had extramedullary (EM) leukemic involvement, and prior to lymphodepletion and CAR 7 had active lesions, a group underrepresented in previous trials. In vivo expansion of CAR T cells was observed in 18 patients. In total 16 patients developed cytokine release syndrome, and 11 patients developed neurotoxicity, with no toxic deaths. All responding patients were referred to an allogeneic hematopoietic stem-cell transplantation. The remission rate was 90%, including resolution of all refractory EM sites. Four responding patients relapsed, 3 who had a PCR-MRD positive remission at 28 days following CAR-T cells and 1 patient 21 months after an MRD-negative response. The estimated 1-year event-free survival and overall survival are 73% and 90%, respectively. Patients with R/R EM ALL may also benefit from CAR-T cell therapy.
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Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Niño , Preescolar , Femenino , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Receptores de Antígenos de Linfocitos T/inmunología , Inducción de Remisión/métodos , Terapia Recuperativa/efectos adversos , Terapia Recuperativa/métodos , Tasa de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Incurable neuroretinal degeneration diseases cause severe vision loss and blindness in millions of patients worldwide. In previous studies, we demonstrated that transplanting human bone marrow stromal cells (hBMSCs) in the extravascular spaces of the choroid (EVSC) of the Royal College of Surgeon rats ameliorated retinal degeneration for up to 5 months. Assessing the safety of hBMSC treatment and graft survival in a large animal is a crucial step before initiating clinical trials. Here, we transplanted hBMSCs into the EVSC compartment of New Zealand White rabbits. No immunosuppressants were used. Transplanted cells were spread across the EVSC covering over 80 percent of the subretinal surface. No cells were detected in the sclera. Cells were retained in the EVSC compartment 10 weeks following transplantation. Spectral domain optical coherence tomography (SD-OCT) and histopathology analysis demonstrated no choroidal hemorrhages, retinal detachment, inflammation, or any untoward pathological reactions in any of transplanted eyes or in the control noninjected contralateral eyes. No reduction in retinal function was recorded by electroretinogram up to 10 weeks following transplantation. This study demonstrates the feasibility and safety of transplanting hBMSCs in the EVSC compartment in a large eye model of rabbits.
RESUMEN
Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28 days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6 weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20 weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Retina/fisiología , Degeneración Retiniana/terapia , Adulto , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Modelos Animales de Enfermedad , Membrana Epirretinal/metabolismo , Membrana Epirretinal/patología , Humanos , Células Madre Mesenquimatosas/metabolismo , Microscopía Fluorescente , Persona de Mediana Edad , Ratas , Trasplante HeterólogoRESUMEN
BACKGROUND: Iron oxide (IO) nanoparticles (NPs) of sizes less than 50 nm are considered to be non-toxic, biodegradable and superparamagnetic. We have previously described the generation of IO NPs coated with Human Serum Albumin (HSA). HSA coating onto the IO NPs enables conjugation of the IO/HSA NPs to various biomolecules including proteins. Here we describe the preparation and characterization of narrow size distribution core-shell NIR fluorescent IO/HSA magnetic NPs conjugated covalently to Fibroblast Growth Factor 2 (FGF2) for biomedical applications. We examined the biological activity of the conjugated FGF2 on human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into bone, cartilage, hepatic, endothelial and neuronal cells and are being studied in clinical trials for treatment of various diseases. FGF2 enhances the proliferation of hBM-MSCs and promotes their differentiation toward neuronal, adipogenic and osteogenic lineages in vitro. RESULTS: The NPs were characterized by transmission electron microscopy, dynamic light scattering, ultraviolet-visible spectroscopy and fluorescence spectroscopy. Covalent conjugation of the FGF2 to the IO/HSA NPs significantly stabilized this growth factor against various enzymes and inhibitors existing in serum and in tissue cultures. IO/HSA NPs conjugated to FGF2 were internalized into hBM-MSCs via endocytosis as confirmed by flow cytometry analysis and Prussian Blue staining. Conjugated FGF2 enhanced the proliferation and clonal expansion capacity of hBM-MSCs, as well as their adipogenic and osteogenic differentiation to a higher extent compared with the free growth factor. Free and conjugated FGF2 promoted the expression of neuronal marker Microtubule-Associated Protein 2 (MAP2) to a similar extent, but conjugated FGF2 was more effective than free FGF2 in promoting the expression of astrocyte marker Glial Fibrillary Acidic Protein (GFAP) in these cells. CONCLUSIONS: These results indicate that stabilization of FGF2 by conjugating the IO/HSA NPs can enhance the biological efficacy of FGF2 and its ability to promote hBM-MSC cell proliferation and trilineage differentiation. This new system may benefit future therapeutic use of hBM-MSCs.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Nanopartículas de Magnetita , Células Madre Mesenquimatosas/citología , Adipogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Estabilidad de Medicamentos , Compuestos Férricos/química , Factor 2 de Crecimiento de Fibroblastos/química , Fluorescencia , Humanos , Nanopartículas de Magnetita/administración & dosificación , Nanopartículas de Magnetita/química , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Tamaño de la Partícula , Fotoblanqueo , Albúmina Sérica/química , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Incurable retinal degenerations affect millions worldwide. Stem cell transplantation rescued visual functions in animal models of retinal degeneration. In those studies, cells were transplanted in subretinal "blebs". A limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection sites. PURPOSE: To develop a minimally-invasive surgical system for stem cell transplantation in the subretina and extravascular spaces of the choroid. METHODS: A novel syringe with flexible needle and adjustable pin was developed. Human bone marrow mesenchymal stem cells [hBM-MSCs) were transplanted in the eyes of RCS rats and NZW rabbits through a longitudinal triangular scleral incision. No immunosuppressants were used. Retinal function was determined by electroretinogram analysis and retinal structure was determined by histological analysis and optical coherence tomography (OCT). RESULTS: Transplanted cells were identified as a thin layer across the subretina and extravascular spaces of the choroid. In RCS rats, cell transplantation delayed photoreceptor degeneration across the entire retina and significantly enhanced retinal functions. No changes in retinal functions were recorded in rabbits following transplantation. No retinal detachment or choroidal hemorrhages were observed. CONCLUSIONS: The novel syringe facilitates cell transplantation across the subretina and extravascular spaces of the choroid using a minimally-invasive procedure. Human BM-MSC transplantation using this system ameliorates retinal degeneration in the animal model. DISCUSSION: This new transplantation system may increase the therapeutic effect of other cell-based therapies and therapeutic agents. This study is expected to lead directly to phase I clinical trials for autologous hBM-MSCs transplantation in patients with retinal degeneration.
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Trasplante de Médula Ósea/métodos , Coroides/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Degeneración Retiniana/cirugía , Animales , Electrorretinografía , Femenino , Humanos , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Conejos , Ratas , Tomografía de Coherencia ÓpticaRESUMEN
Vision incapacitation and blindness associated with retinal degeneration affect millions of people worldwide. Cell based therapy and specifically transplantation of human adult bone marrow-derived stem cells (hBM-MSCs) present possible treatment strategy. Subretinal transplantation of human or rat BM-MSCs was shown previously to improve retinal function in Royal College Surgeons (RCS) rats. In those studies cells were transplanted via a transscleral-transchoroidal approach, creating a localized subretinal bleb. Limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection site. Here we describe a new surgical method for subretinal transplantation that facilitates uniform distribution of transplanted cells as a thin layer along most of the subretinal space. We assessed the therapeutic effect of hBM-MSCs on RCS rats when transplanted either subretinally or intravitreally. We also examined whether a second transplantation can prolong the therapeutic effect. A cell suspension of 2.5 × 10(6) cells in 5 µl was injected subretinally or intravitreally in RCS rats at 28 days postnatal. In the subretinal group, hBM-MSCs were transplanted posterior to the limbus in the superotemporal part of the eye through a longitudinal triangular scleral tunnel reaching the choroid. In the intravitreal group, the cells were injected into the superotemporal part of the vitreous cavity. In cross sections of subretinally transplanted eyes, removed 2 h following transplantation, hBM-MSCs were distributed as a near-homogenous thin layer along most of the subretinal space. In some animals the cells were also detected in the choroid. In the intravitreal injection group, hBM-MSCs were clustered in the vitreous cavity. Transplanted cells could be detected up to 2 weeks after transplantation but not at later time points. Retinal function and structure were assessed by electroretinogram (ERG) and histology analysis, respectively. Six weeks post transplantation, the mean maximal scotopic ERG b-wave amplitude response recorded in RCS control eyes was 1.2 µV. By contrast, in transplanted eyes mean responses of 56.4 µV and 66.2 µV were recorded in the intravitreally and subretinally transplanted eyes, respectively. In the subretinal group, retinal function was significantly higher in transplanted compared with control eyes up to 20 weeks following transplantation. By contrast, in the intravitreal group, rescue of retinal function persisted only up to 12 weeks following transplantation. Histological analysis revealed that 8 weeks following subretinal transplantation, the retinas of control eyes were dystrophic, with outer nuclear layer (ONL) containing a single cell layer. An extensive photoreceptor rescue was demonstrated in transplanted eyes at this time point, with 3-4 cell layers in the ONL along the entire retina. A second subretinal transplantation at 70 days postnatal did not enhance or prolong the therapeutic effect of hBM-MSCs. No immunosuppressants were used and long-term safety analysis demonstrated no gross or microscopic adverse effects. Taken together our findings suggest that transplantation of hBM-MSCs as a thin subretinal layer enhances the therapeutic effect and the safety of cell transplantation.