RESUMEN
The integrin αvß6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvß6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvß6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvß6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvß6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvß6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvß6 in vivo, expanding opportunities for cancer targeting and therapy.
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Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Radiofármacos/metabolismo , Suero/química , Proteínas del Envoltorio Viral/metabolismo , Ciclización , Virus de la Fiebre Aftosa/metabolismo , Humanos , Células K562 , Neoplasias/diagnóstico por imagen , Neoplasias/patologíaRESUMEN
The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.
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Neoplasias , Receptores Quiméricos de Antígenos , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Leucocitos Mononucleares , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores de Transferrina/metabolismo , Linfocitos TRESUMEN
Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivo modified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy's broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.Recognizing that clinical T-cell isolation reagents have high cost and supply constraints, we developed a synthetic DNA aptamer and complementary reversal agent technology that isolates label-free CD8+ T cells with high purity and yield from peripheral blood mononuclear cells. Encouragingly, CAR T cells manufactured from both antibody- and aptamer-isolated T cells were comparable in therapeutic potency. Discovery and design of other T-cell specific aptamers and corresponding reversal reagents could fully realize the potential of this approach, enabling inexpensive isolation of multiple distinct T-cell populations in a single isolation step.Current ex vivo T-cell activation materials do not accurately mimic in situ T-cell activation by antigen presenting cells (APCs). They cause unequal CD4+ and CD8+ T-cell expansion, necessitating separate production of CD4+ and CD8+ CAR T cells for therapies that call for balanced infusion compositions. To address these shortcomings, we designed a panel of biodegradable cell-templated silica microparticles with supported lipid bilayers that display stimulatory ligands for T-cell activation. High membrane fluidity, elongated shape, and rough surface topography, all properties of endogenous APCs, were found to be favorable parameters for activation, promoting unbiased and efficient CD4/CD8 T-cell expansion while not terminally differentiating the cells.Viral and electroporation-based gene delivery systems have various drawbacks. Viral vectors are expensive and have limited cargo sizes, whereas electroporation is highly cytotoxic. Thus, low-cost nonviral platforms that transfect T cells with low cytotoxicity and high efficiency are needed for CAR gene delivery. Our group thus synthesized a panel of cationic polymers with different architectures and evaluated their T-cell transfection ability. We identified a comb-shaped polymer formulation that transfected primary T cells with low cytotoxicity, although transfection efficiency was low compared to conventional methods. Analysis of intracellular and extracellular barriers to transfection revealed low uptake of polyplexes and high endosomal pH in T cells, alluding to biological and polymer properties that could be further improved.These innovations represent just a few recent developments in the biomaterials field for addressing CAR T-cell production needs. Together, these technologies and their future advancement will pave the way for economical and straightforward CAR T-cell manufacturing.
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Materiales Biocompatibles/química , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Materiales Biocompatibles/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Técnicas de Transferencia de Gen , Humanos , Separación Inmunomagnética/métodos , Inmunoterapia Adoptiva , Nanoestructuras/química , Neoplasias/terapia , Polímeros/química , Receptores Quiméricos de Antígenos/genética , Dióxido de Silicio/químicaRESUMEN
Immunotherapy with the adoptive transfer of T cells redirected with CD19-specific chimeric antigen receptors (CARs) for B-lineage acute lymphoblastic leukemia (ALL) can salvage >80% of patients having relapsed/refractory disease. The therapeutic index of this emerging modality is attenuated by the occurrence of immunologic toxicity syndromes that occur upon CAR T-cell engraftment. Here, we report on the low incidence of severe cytokine release syndrome (CRS) in a subject treated with a CAR T-cell product composed of a defined ratio CD4:CD8 T-cell composition with a 4-1BB:zeta CAR targeting CD19 who also recieved early intervention treatment. We report that early intervention with tocilizumab and/or corticosteroids may reduce the frequency at which subjects transition from mild CRS to severe CRS. Although early intervention doubled the numbers of subjects dosed with tocilizumab and/or corticosteroids, there was no apparent detrimental effect on minimal residual disease-negative complete remission rates or subsequent persistence of functional CAR T cells compared with subjects who did not receive intervention. Moreover, early intervention therapy did not increase the proportion of subjects who experience neurotoxicity or place subjects at risk for infectious sequelae. These data support the contention that early intervention with tocilizumab and/or corticosteroids in subjects with early signs of CRS is without negative impact on the antitumor potency of CD19 CAR T cells. This intervention serves to enhance the therapeutic index in relapsed/refractory patients and provides the rationale to apply CAR T-cell therapy more broadly in ALL therapy. This trial was registered at www.clinicaltrials.gov as #NCT020284.
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Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Síndrome de Liberación de Citoquinas/etiología , Inmunoterapia Adoptiva , Leucemia-Linfoma Linfoblástico de Células Precursoras B/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Adolescente , Corticoesteroides/administración & dosificación , Corticoesteroides/farmacología , Adulto , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Niño , Preescolar , Síndrome de Liberación de Citoquinas/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Incidencia , Lactante , Masculino , Clasificación del Tumor , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Adulto JovenRESUMEN
OBJECTIVE: To test whether systemic cytokine release is associated with central nervous system inflammatory responses and glial injury in immune effector cell-associated neurotoxicity syndrome (ICANS) after chimeric antigen receptor (CAR)-T cell therapy in children and young adults. METHODS: We performed a prospective cohort study of clinical manifestations as well as imaging, pathology, CSF, and blood biomarkers on 43 subjects ages 1 to 25 who received CD19-directed CAR/T cells for acute lymphoblastic leukemia (ALL). RESULTS: Neurotoxicity occurred in 19 of 43 (44%) subjects. Nine subjects (21%) had CTCAE grade 3 or 4 neurological symptoms, with no neurotoxicity-related deaths. Reversible delirium, headache, decreased level of consciousness, tremor, and seizures were most commonly observed. Cornell Assessment of Pediatric Delirium (CAPD) scores ≥9 had 94% sensitivity and 33% specificity for grade ≥3 neurotoxicity, and 91% sensitivity and 72% specificity for grade ≥2 neurotoxicity. Neurotoxicity correlated with severity of cytokine release syndrome, abnormal past brain magnetic resonance imaging (MRI), and higher peak CAR-T cell numbers in blood, but not cerebrospinal fluid (CSF). CSF levels of S100 calcium-binding protein B and glial fibrillary acidic protein increased during neurotoxicity, indicating astrocyte injury. There were concomitant increases in CSF white blood cells, protein, interferon-γ (IFNγ), interleukin (IL)-6, IL-10, and granzyme B (GzB), with concurrent elevation of serum IFNγ IL-10, GzB, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein 1 alpha, and tumor necrosis factor alpha, but not IL-6. We did not find direct evidence of endothelial activation. INTERPRETATION: Our data are most consistent with ICANS as a syndrome of systemic inflammation, which affects the brain through compromise of the neurovascular unit and astrocyte injury. ANN NEUROL 2019.
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Antígenos CD19/efectos adversos , Inmunoterapia Adoptiva/efectos adversos , Neuroglía/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico por imagen , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Antígenos CD19/administración & dosificación , Antígenos CD19/inmunología , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Inmunoterapia Adoptiva/tendencias , Lactante , Masculino , Neuroglía/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Estudios Prospectivos , Adulto JovenRESUMEN
Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of naïve and central memory precursors. Significant numbers of Ki67+/PD1+ T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112â¯×â¯106 T cells were amenable to immunomagnetic selection, CD3â¯×â¯28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR+ CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.
Asunto(s)
Traslado Adoptivo , Criopreservación , Movilización de Célula Madre Hematopoyética , Neuroblastoma , Células Madre de Sangre Periférica , Receptores Quiméricos de Antígenos/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neuroblastoma/inmunología , Neuroblastoma/patología , Neuroblastoma/terapia , Células Madre de Sangre Periférica/inmunología , Células Madre de Sangre Periférica/patología , Estudios Retrospectivos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A patient with recurrent multifocal glioblastoma received chimeric antigen receptor (CAR)-engineered T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2). Multiple infusions of CAR T cells were administered over 220 days through two intracranial delivery routes - infusions into the resected tumor cavity followed by infusions into the ventricular system. Intracranial infusions of IL13Rα2-targeted CAR T cells were not associated with any toxic effects of grade 3 or higher. After CAR T-cell treatment, regression of all intracranial and spinal tumors was observed, along with corresponding increases in levels of cytokines and immune cells in the cerebrospinal fluid. This clinical response continued for 7.5 months after the initiation of CAR T-cell therapy. (Funded by Gateway for Cancer Research and others; ClinicalTrials.gov number, NCT02208362 .).
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Glioblastoma/terapia , Inmunoterapia Adoptiva , Recurrencia Local de Neoplasia/terapia , Receptores de Antígenos de Linfocitos T/uso terapéutico , Ingeniería Celular , Terapia Combinada , Humanos , Subunidad alfa2 del Receptor de Interleucina-13 , Masculino , Persona de Mediana EdadRESUMEN
Transitioning CD19-directed chimeric antigen receptor (CAR) T cells from early-phase trials in relapsed patients to a viable therapeutic approach with predictable efficacy and low toxicity for broad application among patients with high unmet need is currently complicated by product heterogeneity resulting from transduction of undefined T-cell mixtures, variability of transgene expression, and terminal differentiation of cells at the end of culture. A phase 1 trial of 45 children and young adults with relapsed or refractory B-lineage acute lymphoblastic leukemia was conducted using a CD19 CAR product of defined CD4/CD8 composition, uniform CAR expression, and limited effector differentiation. Products meeting all defined specifications occurred in 93% of enrolled patients. The maximum tolerated dose was 106 CAR T cells per kg, and there were no deaths or instances of cerebral edema attributable to product toxicity. The overall intent-to-treat minimal residual disease-negative (MRD-) remission rate for this phase 1 study was 89%. The MRD- remission rate was 93% in patients who received a CAR T-cell product and 100% in the subset of patients who received fludarabine and cyclophosphamide lymphodepletion. Twenty-three percent of patients developed reversible severe cytokine release syndrome and/or reversible severe neurotoxicity. These data demonstrate that manufacturing a defined-composition CD19 CAR T cell identifies an optimal cell dose with highly potent antitumor activity and a tolerable adverse effect profile in a cohort of patients with an otherwise poor prognosis. This trial was registered at www.clinicaltrials.gov as #NCT02028455.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/trasplante , Recurrencia Local de Neoplasia/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Adolescente , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Recurrencia Local de Neoplasia/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Inducción de Remisión , Adulto JovenRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.
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Tratamiento Basado en Trasplante de Células y Tejidos , Gangliósidos/inmunología , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Receptores Quiméricos de Antígenos , Retinoblastoma/terapia , Linfocitos T/metabolismo , Línea Celular Tumoral , Niño , Preescolar , Citotoxicidad Inmunológica , Femenino , Humanos , Lactante , Masculino , Retinoblastoma/inmunología , Retinoblastoma/metabolismo , Estudios Retrospectivos , Linfocitos T/inmunologíaRESUMEN
The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have â¼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.
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Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Animales , Femenino , Humanos , Inmunoterapia Adoptiva , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy.
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Antígenos CD19/genética , N-Metiltransferasa de Histona-Lisina/genética , Inmunoterapia Adoptiva , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Escape del Tumor , Antígenos CD19/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Cromosomas Humanos Par 11/genética , Células Clonales , Terapia Combinada , Femenino , Humanos , Inmunofenotipificación , Lactante , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Depleción Linfocítica , Persona de Mediana Edad , Células Madre Neoplásicas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Recurrencia , Inducción de Remisión , Terapia Recuperativa , Translocación GenéticaRESUMEN
Myeloablative autologous hematopoietic stem cell transplantation (HSCT) is a mainstay of therapy for relapsed intermediate-grade B-cell non-Hodgkin lymphoma (NHL); however, relapse rates are high. In phase 1 studies designed to improve long-term remission rates, we administered adoptive T-cell immunotherapy after HSCT, using ex vivo-expanded autologous central memory-enriched T cells (TCM) transduced with lentivirus expressing CD19-specific chimeric antigen receptors (CARs). We present results from 2 safety/feasibility studies, NHL1 and NHL2, investigating different T-cell populations and CAR constructs. Engineered TCM-derived CD19 CAR T cells were infused 2 days after HSCT at doses of 25 to 200 × 10(6) in a single infusion. In NHL1, 8 patients safely received T-cell products engineered from enriched CD8(+) TCM subsets, expressing a first-generation CD19 CAR containing only the CD3ζ endodomain (CD19R:ζ). Four of 8 patients (50%; 95% confidence interval [CI]: 16-84%) were progression free at both 1 and 2 years. In NHL2, 8 patients safely received T-cell products engineered from enriched CD4(+) and CD8(+) TCM subsets and expressing a second-generation CD19 CAR containing the CD28 and CD3ζ endodomains (CD19R:28ζ). Six of 8 patients (75%; 95% CI: 35-97%) were progression free at 1 year. The CD4(+)/CD8(+) TCM-derived CD19 CAR T cells (NHL2) exhibited improvement in expansion; however, persistence was ≤28 days, similar to that seen by others using CD28 CARs. Neither cytokine release syndrome nor delayed hematopoietic engraftment was observed in either trial. These data demonstrate the safety and feasibility of CD19 CAR TCM therapy after HSCT. Trials were registered at www.clinicaltrials.gov as #NCT01318317 and #NCT01815749.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Memoria Inmunológica , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Linfocitos T/trasplante , Adulto , Anciano , Antígenos CD19/metabolismo , Recuento de Células , Terapia Combinada/efectos adversos , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Linfoma de Células B/inmunología , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/terapia , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Trasplante Autólogo , Adulto JovenRESUMEN
A major advance in adoptive T-cell therapy (ACT) is the ability to efficiently endow patient's T cells with reactivity for tumor antigens through the stable or regulated introduction of genes that encode high affinity tumor-targeting T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). Case reports and small series of patients treated with TCR- or CAR-modified T cells have shown durable responses in a subset of patients, particularly with B-cell malignancies treated with T cells modified to express a CAR that targets the CD19 molecule. However, many patients do not respond to therapy and serious on and off-target toxicities have been observed with TCR- and CAR-modified T cells. Thus, challenges remain to make ACT with gene-modified T cells a reproducibly effective and safe therapy and to expand the breadth of patients that can be treated to include those with common epithelial malignancies. This review discusses research topics in our laboratories that focus on the design and implementation of ACT with CAR-modified T cells. These include cell intrinsic properties of distinct T-cell subsets that may facilitate preparing therapeutic T-cell products of defined composition for reproducible efficacy and safety, the design of tumor targeting receptors that optimize signaling of T-cell effector functions and facilitate tracking of migration of CAR-modified T cells in vivo, and novel CAR designs that have alternative ligand binding domains or confer regulated function and/or survival of transduced T cells.
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Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos/genética , Antígenos/inmunología , Humanos , Inmunoterapia Adoptiva/métodos , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Proteínas Mutantes , Linfocitos T/metabolismo , Proteínas Virales/metabolismo , Dependovirus/genética , Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Recombinación Homóloga , Humanos , ARN Guía de Kinetoplastida/genética , Transducción Genética , Proteínas Virales/genéticaRESUMEN
Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.
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Sistemas CRISPR-Cas , Endonucleasas , Edición Génica , Receptores de Antígenos de Linfocitos T/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Sitios de Unión , Técnicas de Cultivo de Célula , Línea Celular , Marcación de Gen , Técnicas de Transferencia de Gen , Sitios Genéticos , Genoma , Humanos , Inmunofenotipificación , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Fenotipo , Unión Proteica , Proteínas Recombinantes de Fusión , Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.
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Bacteriófagos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Macrófagos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Algoritmos , Animales , Células Cultivadas , Ligandos , Macrófagos/citología , Ratones , Fragmentos de Péptidos/genéticaRESUMEN
Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.
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Citotoxicidad Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Mieloide/inmunología , Receptores de Antígenos/inmunología , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Enfermedad Aguda , Animales , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Células HEK293 , Humanos , Inmunoterapia Adoptiva/métodos , Subunidad alfa del Receptor de Interleucina-3/genética , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Células K562 , Leucemia Mieloide/patología , Leucemia Mieloide/terapia , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Linfocitos T/metabolismo , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (â¼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications.
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Ingeniería Celular/métodos , Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Metotrexato/metabolismo , Selección Genética , Linfocitos T/fisiología , Expresión Génica , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , TransgenesRESUMEN
The adoptive transfer of donor T cells that have been genetically modified to recognize leukemia could prevent or treat leukemia relapse after allogeneic HSCT (allo-HSCT). However, adoptive therapy after allo-HSCT should be performed with T cells that have a defined endogenous TCR specificity to avoid GVHD. Ideally, T cells selected for genetic modification would also have the capacity to persist in vivo to ensure leukemia eradication. Here, we provide a strategy for deriving virus-specific T cells from CD45RA(-)CD62L(+)CD8(+) central memory T (T(CM)) cells purified from donor blood with clinical grade reagents, and redirect their specificity to the B-cell lineage marker CD19 through lentiviral transfer of a gene encoding a CD19-chimeric Ag receptor (CAR). Virus-specific T(CM) were selectively transduced by exposure to the CD19 CAR lentivirus after peptide stimulation, and bi-specific cells were subsequently enriched to high purity using MHC streptamers. Activation of bi-specific T cells through the CAR or the virus-specific TCR elicited phosphorylation of downstream signaling molecules with similar kinetics, and induced comparable cytokine secretion, proliferation, and lytic activity. These studies identify a strategy for tumor-specific therapy with CAR-modified T cells after allo-HSCT, and for comparative studies of CAR and TCR signaling.
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Antígenos CD19/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Lentivirus/inmunología , Leucemia/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos/inmunología , Traslado Adoptivo , Antígenos CD19/genética , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citometría de Flujo , Humanos , Inmunoterapia Adoptiva , Lentivirus/genética , Leucemia/inmunología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , Receptores de Antígenos/genética , Receptores de Antígenos de Linfocitos T/genética , Células Tumorales CultivadasRESUMEN
Cellular immune responses have the potential to elicit dramatic and sustained clinical remissions in lymphoma patients. Recent clinical trial data demonstrate that modification of T cells with chimeric antigen receptors (CARs) is a promising strategy. T cells containing CARs with costimulatory domains exhibit improved activity against tumors. We conducted a pilot clinical trial testing a "third-generation" CD20-specific CAR with CD28 and 4-1BB costimulatory domains in patients with relapsed indolent B-cell and mantle cell lymphomas. Four patients were enrolled, and 3 received T-cell infusions after cyclophosphamide lymphodepletion. Treatment was well tolerated, although one patient developed transient infusional symptoms. Two patients without evaluable disease remained progression-free for 12 and 24 months. The third patient had an objective partial remission and relapsed at 12 months after infusions. Modified T cells were detected by quantitative PCR at tumor sites and up to 1 year in peripheral blood, albeit at low levels. No evidence of host immune responses against infused cells was detected. In conclusion, adoptive immunotherapy with CD20-specific T cells was well tolerated and was associated with antitumor activity. We will pursue alternative gene transfer technologies and culture conditions in future studies to improve CAR expression and cell production efficiency.