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PURPOSE: T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). METHODS: Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. RESULTS: Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. CONCLUSION: Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.
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Neoplasias del Sistema Nervioso Central , Gangliósidos , Receptores Quiméricos de Antígenos , Humanos , Niño , Adolescente , Preescolar , Masculino , Femenino , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Lactante , Adulto Joven , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/inmunología , Neoplasias del Sistema Nervioso Central/terapia , Gangliósidos/inmunología , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Clasificación del Tumor , Glioma/tratamiento farmacológico , Glioma/patología , Glioma/terapia , Glioma/inmunología , Subunidad alfa del Receptor de Interleucina-7RESUMEN
Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.
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PURPOSE: A phase 2 study of stereotactic body radiation therapy (SBRT) and in situ oncolytic virus therapy in metastatic non-small cell lung cancer (mNSCLC) followed by pembrolizumab (STOMP) was designed to explore the dual approach in enhancing single pembrolizumab with ADV/HSV-tk plus valacyclovir gene therapy and SBRT in mNSCLC. METHODS AND MATERIALS: STOMP is a single-arm, open-label phase 2 study. Patients with mNSCLC received intratumoral injections of ADV/HSV-tk (5 × 1011 vp) and SBRT (30 Gy in 5 fractions) followed by pembrolizumab 200 mg IV every 3 weeks until disease progression or intolerable toxicity. The primary endpoint was overall response rate (ORR) (complete response [CR] and partial response [PR]). Secondary endpoints included clinical benefit rate (CBR) (CR, PR and stable disease [SD]), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: 28 patients were enrolled, of whom 27 were evaluated for response. The ORR was 33.3%, including 2 CR (7.4%) and 7 PR (25.9%). CBR was 70.4%. Six of eight (75.0%) patients who were immune checkpoint inhibitor (ICI) refractory derived clinical benefits. Responders had durable responses with median PFS, and OS not reached. The entire cohort had a median PFS of 7.4 months (95% CI, 5.1-9.6 months), and median OS of 18.1 months (95% CI, 15.4-20.9 months). The combination was well tolerated, with grade 3 or higher toxicity in 6 (21.4%) patients. CONCLUSIONS: The dual approach of in situ ADV/HSV-tk plus valacyclovir gene therapy and SBRT as a chemotherapy-sparing strategy to enhance the antitumor effect of pembrolizumab is a well-tolerated encouraging treatment in patients with mNSCLC.
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Anticuerpos Monoclonales Humanizados , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Viroterapia Oncolítica , Radiocirugia , Humanos , Radiocirugia/efectos adversos , Viroterapia Oncolítica/efectos adversos , Valaciclovir/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
ABSTRACT: Despite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.
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Inmunoterapia Adoptiva , Linfoma de Células T , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfocitos T , Enfermedad Crónica , Linfoma de Células T/tratamiento farmacológico , Antígenos CD19RESUMEN
BACKGROUND AIMS: Stem and progenitor cells of hematopoietic and mesenchymal lineages reside in the bone marrow under low oxygen (O2) saturation. O2 levels used in ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) affect proliferation, metabolism and differentiation. METHODS: Using cell-based assays and transcriptome and proteome data, the authors compared MSC cultures simultaneously grown under a conventional 19.95% O2 atmosphere or at 5% O2. RESULTS: In 5% O2, MSCs showed better proliferation and higher self-renewal ability, most probably sustained by enhanced signaling activity of mitogen-activated protein kinase and mammalian target of rapamycin pathways. Non-oxidative glycolysis-based energy metabolism supported growth and proliferation in 5% O2 cultures, whereas MSCs grown under 19.95% O2 also utilized oxidative phosphorylation. Cytoprotection mechanisms used by cells under 5% O2 differed from 19.95% O2 suggesting differences in the triggers of cell stress between these two O2 conditions. CONCLUSIONS: Based on the potential benefits for the growth and metabolism of MSCs, the authors propose the use of 5% O2 for MSC culture.
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Proteínas Quinasas Activadas por Mitógenos , Oxígeno , Oxígeno/metabolismo , Células Cultivadas , Sirolimus , Proliferación Celular , Diferenciación Celular/fisiología , Serina-Treonina Quinasas TORRESUMEN
PURPOSE: A Phase 2 trial of stereotactic radiotherapy and in situ cytotoxic virus therapy in patients with metastatic triple-negative breast cancer (mTNBC) followed by pembrolizumab (STOMP) was designed to evaluate dual approach of enhancing single-agent immune checkpoint blockade with adenovirus-mediated expression of herpes-simplex-virus thymidine-kinase (ADV/HSV-tk) plus valacyclovir gene therapy and stereotactic body radiotherapy (SBRT) in patients with mTNBC. PATIENTS AND METHODS: In this single-arm, open-label Phase 2 trial, patients with mTNBC were treated with ADV/HSV-tk [5 × 1011 virus particles (vp)] intratumoral injection, followed by SBRT to the injected tumor site, then pembrolizumab (200 mg, every 3 weeks). The primary endpoint was clinical benefit rate [CBR; complete response (CR), partial response (PR), or stable disease (SD) ≥ 24 weeks per RECIST version1.1 at non-irradiated site]. Secondary endpoints included duration on treatment (DoT), overall survival (OS), and safety. Exploratory endpoints included immune response to treatment assessed by correlative tissue and blood-based biomarkers. RESULTS: Twenty-eight patients were enrolled and treated. CBR was seen in 6 patients (21.4%), including 2 CR (7.1%), 1 PR (3.6%), and 3 SD (10.7%). Patients with clinical benefit had durable responses, with median DoT of 9.6 months and OS of 14.7 months. The median OS was 6.6 months in the total population. The combination was well tolerated. Correlative studies with Cytometry by Time of Flight (CyTOF) and imaging mass cytometry (IMC) revealed a significant increase of CD8 T cells in responders and of myeloid cells in non-responders. CONCLUSIONS: The median OS increased by more than 2-fold in patients with clinical benefit. The therapy is a well-tolerated treatment in heavily pretreated patients with mTNBC. Early detection of increased effector and effector memory CD8 T cells and myeloids correlate with response and non-response, respectively.
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Radiocirugia , Neoplasias de la Mama Triple Negativas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Genética , Humanos , Inhibidores de Puntos de Control Inmunológico , Timidina/uso terapéutico , Timidina Quinasa/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Valaciclovir/uso terapéuticoRESUMEN
Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy.
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Antígenos CD19/inmunología , Inmunoterapia Adoptiva , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Anciano , Terapia Combinada , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Linfoma no Hodgkin/diagnóstico , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T/metabolismo , Trasplante Autólogo , Resultado del TratamientoRESUMEN
BACKGROUND AIMS: Activated NK cells (aNK) generated by expansion of a human interleukin-2-dependent NK cell line (NK-92) were shown to mediate strong anti-leukemia activity. This phase 1 study evaluated feasibility, safety, and activity of aNK cells adoptively transferred to patients with refractory/relapsed acute myeloid leukemia (AML). In addition, effects of these aNK cells on the patient's immune system were evaluated. METHODS: Two cell-dose levels (1 × 109 cells/m2 and 3 × 109 cells/m2) were used. One treatment course consisted of two infusions of the same cell dose, each cell infusion delivered 24 h apart. The aNK cells were administered in the outpatient setting. RESULTS: Seven patients with refractory/relapsed AML were treated with a total of 20 aNK cell infusions. None of the 7 patients experienced dose-limiting toxicities during the aNK cell administration or during 21 days of the post-infusion observation period. No grade 3-4 toxicities (probable or definite) related to aNK cell infusions occurred. Activity was transient in 3 of 7 patients. No significant changes in the patient's lymphocyte counts, subsets frequency, phenotype or activity were observed post-infusion. Cell dose-dependent effects in the plasma levels of several cytokines were observed. DISCUSSION: The trial demonstrated the safety and feasibility of adoptive cell therapy with "off-the-shelf" aNK cells in patients with refractory/relapsed AML. These data provide the foundation for future combination immunotherapy trials and for the optimization of aNK cell based therapies in patients with AML.
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Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/trasplante , Leucemia Mieloide Aguda/terapia , Anciano , Anciano de 80 o más Años , Trasplante de Células/efectos adversos , Trasplante de Células/métodos , Citocinas/sangre , Femenino , Humanos , Inmunoterapia Adoptiva/efectos adversos , Interleucina-2/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.
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BACKGROUND: Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity. METHODS: We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab. RESULTS: No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity. CONCLUSION: CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT01316146. FUNDING: National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).
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Enfermedad de Hodgkin/terapia , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/terapia , Receptores de Antígenos de Linfocitos T/química , Linfocitos T/citología , Adulto , Antineoplásicos/química , Brentuximab Vedotina , Antígenos CD28/química , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Enfermedad de Hodgkin/inmunología , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/uso terapéutico , Inmunofenotipificación , Linfoma Anaplásico de Células Grandes/inmunología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Recurrencia Local de Neoplasia , Acondicionamiento Pretrasplante , Resultado del Tratamiento , Adulto JovenRESUMEN
Targeting disialoganglioside (GD2) on neuroblastoma (NB) with T cells expressing a first-generation chimeric antigen receptor (CAR) was safe, but the cells had poor expansion and long-term persistence. We developed a third-generation GD2-CAR (GD2-CAR3) and hypothesized that GD2-CAR3 T cells (CARTs) would be safe and effective. This phase 1 study enrolled relapsed or refractory NB patients in three cohorts. Cohort 1 received CART alone, cohort 2 received CARTs plus cyclophosphamide and fludarabine (Cy/Flu), and cohort 3 was treated with CARTs, Cy/Flu, and a programmed death-1 (PD-1) inhibitor. Eleven patients were treated with CARTs. The infusions were safe, and no dose-limiting toxicities occurred. CARTs were detectable in cohort 1, but the lymphodepletion induced by Cy/Flu increased circulating levels of the homeostatic cytokine interleukin (IL)-15 (p = 0.003) and increased CART expansion by up to 3 logs (p = 0.03). PD-1 inhibition did not further enhance expansion or persistence. Antitumor responses at 6 weeks were modest. We observed a striking expansion of CD45/CD33/CD11b/CD163+ myeloid cells (change from baseline, p = 0.0126) in all patients, which may have contributed to the modest early antitumor responses; the effect of these cells merits further study. Thus, CARTs are safe, and Cy/Flu can further increase their expansion.
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Antineoplásicos Inmunológicos/uso terapéutico , Inmunoterapia Adoptiva , Neuroblastoma/inmunología , Neuroblastoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adolescente , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Niño , Preescolar , Estudios de Cohortes , Terapia Combinada , Citocinas/sangre , Femenino , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Recuento de Linfocitos , Depleción Linfocítica , Masculino , Terapia Molecular Dirigida , Células Mieloides/metabolismo , Neuroblastoma/mortalidad , Neuroblastoma/patología , Receptores de Antígenos de Linfocitos T/genética , Acondicionamiento Pretrasplante , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND AIMS: Extensive animal data indicate that mesenchymal stromal cells (MSCs) improve outcome in stroke models. Intra-arterial (IA) injection is a promising route of delivery for MSCs. Therapeutic effect of MSCs in stroke is likely based on the broad repertoire of secreted trophic and immunomodulatory cytokines produced by MSCs. We determined the differential effects of exposing MSCs to different types of clinically relevant vehicles, and/or different additives and passage through a catheter relevant to IA injections. METHODS: MSCs derived from human bone marrow were tested in the following vehicles: 5% albumin (ALB), 6% Hextend (HEX) and 40% dextran (DEX). Each solution was tested (i) alone, (ii) with low-dose heparin, (iii) with 10% Omnipaque, or (iv) a combination of heparin and Omnipaque. Cells in vehicles were collected directly or passed through an IA catheter, and MSC viability and cytokine release profiles were assessed. RESULTS: Cell viability remained above 90% under all tested conditions with albumin being the highest at 97%. Viability was slightly reduced after catheter passage or exposure to heparin or Omnipaque. Catheter passage had little effect on MSC cytokine secretion. ALB led to increased release of angiogenic factors such as vascular endothelial growth factor compared with other vehicles, while HEX and DEX led to suppression of pro-inflammatory cytokines such as interleukin-6. However, when these three vehicles were subjected to catheter passage and/or exposure to additives, the cytokine release profile varied depending on the combination of conditions to which MSCs were exposed. DISCUSSION: Exposure of MSCs to certain types of vehicles or additives changes the profile of cytokine secretion. The activation phenotype of MSCs may therefore be affected by the vehicles used for these cells or the exposure to the adjuvants used in their administration.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Dispositivos de Acceso Vascular , Adyuvantes Inmunológicos/farmacología , Células de la Médula Ósea/citología , Supervivencia Celular , Citocinas/metabolismo , Heparina/farmacología , Humanos , Interleucina-6/metabolismo , Yohexol/farmacología , Trasplante de Células Madre Mesenquimatosas/instrumentación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Suspensiones , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS: We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS: κ.CART expansion peaked 1-2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2-17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION: κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00881920. FUNDING: National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018.
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Cadenas kappa de Inmunoglobulina/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma no Hodgkin/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Adulto , Anciano , Antígenos CD19/inmunología , Ensayo de Inmunoadsorción Enzimática , Estudios de Factibilidad , Femenino , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma no Hodgkin/inmunología , Masculino , Persona de Mediana Edad , Inducción de Remisión , Retroviridae/metabolismo , Resultado del TratamientoRESUMEN
PURPOSE: The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. PATIENTS AND METHODS: We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). RESULTS: We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). CONCLUSION: This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence.
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Neoplasias Óseas/terapia , Inmunoterapia/métodos , Receptor ErbB-2/metabolismo , Sarcoma/terapia , Linfocitos T/inmunología , Adolescente , Adulto , Neoplasias Óseas/metabolismo , Niño , Femenino , Humanos , Estimación de Kaplan-Meier , Imagen por Resonancia Magnética , Masculino , Dosis Máxima Tolerada , Metástasis de la Neoplasia , Tumores Neuroectodérmicos/metabolismo , Tumores Neuroectodérmicos/terapia , Osteosarcoma/metabolismo , Osteosarcoma/terapia , Tomografía de Emisión de Positrones , Receptor ErbB-2/genética , Receptores de Antígenos de Linfocitos T/química , Recurrencia , Sarcoma/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/terapia , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS: We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS: After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS: The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.
Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Mesenquimatosas/citología , Animales , Reactores Biológicos , Diferenciación Celular/genética , Linaje de la Célula , Humanos , Trasplante de Células Madre Mesenquimatosas , RatasRESUMEN
Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/inmunología , Trasplante de Células Madre Hematopoyéticas , Leucemia Linfocítica Crónica de Células B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/trasplante , Adenoviridae/inmunología , Adulto , Antígenos CD19/genética , Antineoplásicos/uso terapéutico , Linfocitos B/patología , Niño , Citomegalovirus/inmunología , Femenino , Expresión Génica , Herpesvirus Humano 4/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/virología , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/virología , Ingeniería de Proteínas , Recurrencia , Linfocitos T/inmunología , Linfocitos T/virología , Trasplante HomólogoRESUMEN
Mesenchymal stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytes, osteoblasts and chondroblasts as well as secreting a vast array of soluble mediators. This potentially makes MSCs important mediators of a variety of therapeutic applications. They are actively under evaluation for immunomodulatory purposes such as graft-versus-host disease and Crohn's disease as well as regenerative applications such as stroke and congestive heart failure. We report our method of generating clinical-grade MSCs together with suggestions gathered from manufacturing experience in our Good Manufacturing Practices facility.
Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Leucocitos Mononucleares/citología , Células Madre Mesenquimatosas/citología , Diferenciación Celular , Células Cultivadas , Ensayos Clínicos Fase I como Asunto , Criopreservación , Humanos , Trasplante de Células Madre Mesenquimatosas/métodosRESUMEN
BACKGROUND AIMS: Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. METHODS: We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). RESULTS: Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. CONCLUSIONS: We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva , Células K562/citología , Células Asesinas Naturales/citología , Linfocitos T , Ligando 4-1BB/metabolismo , Eliminación de Componentes Sanguíneos , Técnicas de Cultivo de Célula , Supervivencia Celular , Criopreservación , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.
Asunto(s)
Traslado Adoptivo/métodos , Neoplasias Óseas/terapia , Gangliósidos/inmunología , Terapia Genética/métodos , Neuroblastoma/terapia , Receptores de Antígenos de Linfocitos T/genética , Adolescente , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/inmunología , Neoplasias de la Médula Ósea/terapia , Neoplasias Óseas/genética , Neoplasias Óseas/inmunología , Línea Celular Tumoral , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Memoria Inmunológica/inmunología , Masculino , Neoplasia Residual/genética , Neoplasia Residual/inmunología , Neoplasia Residual/terapia , Neuroblastoma/genética , Neuroblastoma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto JovenRESUMEN
Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.