RESUMEN
Several CD19-targeting CAR-T cells are used to treat leukemias and lymphomas; however, relapsed and/or refractory (R/R) disease is still observed in a significant number of patients. Additionally, the success of CD19-CAR-T cell therapies is not uniform across hematological malignancies, particularly in chronic lymphocytic leukemia (CLL). In this study, we present the development of a novel CAR-T cell therapy targeting B-cell activating factor receptor (BAFF-R), a key regulator of B-cell proliferation and maturation. A new monoclonal antibody against BAFF-R was generated from a hybridoma clone and used to create a novel MC10029 CAR construct. Through a series of in vitro and in vivo models using the Nalm-6 cell line for leukemia and the Z138 cell line for lymphoma, we demonstrated the antigen-specific cytotoxicity of MC10029 CAR-T cells against tumor cells. Additionally, MC10029 CAR-T cells exhibited potent antitumor effects against CD19 knockout tumor cells, mimicking CD19-negative R/R disease. MC10029 CAR-T cells were specifically targeted to CLL, in which BAFF-R is nearly always expressed. The cytotoxicity of MC10029 CAR-T cells was first shown in the MEC-1 CLL cell line, before we turned our efforts to subject-derived samples. Using healthy donor-engineered MC10029 CAR-T cells against enriched primary tumor cells, followed by subject-derived MC10029 CAR-T cells against autologous tumor cells, we showed the efficacy of MC10029 CAR-T cells against CLL subject samples. With these robust data, we have advanced to the production of MC10029 CAR-T cells, using GMP lentivirus, and obtained an IND approval in preparation for a Phase 1 clinical trial.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Leucemia , Linfoma , Receptores Quiméricos de Antígenos , Humanos , Leucemia Linfocítica Crónica de Células B/terapia , Inmunoterapia Adoptiva , Linfoma/terapia , Antígenos CD19RESUMEN
Nepafenac ophthalmic suspensions, 0.1% (NEVANAC(®)) and 0.3% (ILEVRO™), are topical nonsteroidal anti-inflammatory drug (NSAID) products approved in the United States, Europe and various other countries to treat pain and inflammation associated with cataract surgery. NEVANAC is also approved in Europe for the reduction in the risk of postoperative macular edema (ME) associated with cataract surgery in diabetic patients. The efficacy against ME suggests that topical administration leads to distribution of nepafenac or its active metabolite amfenac to the posterior segment of the eye. This article evaluates the ocular distribution of nepafenac and amfenac and the extent of local delivery to the posterior segment of the eye, following topical ocular instillation in animal models. Nepafenac ophthalmic suspension was instilled unilaterally in New Zealand White rabbits as either a single dose (0.1%; one drop) or as multiple doses (0.3%, one drop, once-daily for 4 days, or 0.1% one drop, three-times daily for 3 days and one morning dose on day 4). Nepafenac (0.3%) was also instilled unilaterally in cynomolgus monkeys as multiple doses (one drop, three-times daily for 7 days). Nepafenac and amfenac concentrations in harvested ocular tissues were measured using high-performance liquid chromatography/mass spectrometry. Locally-distributed compound concentrations were determined as the difference in levels between dosed and undosed eyes. In single-dosed rabbit eyes, peak concentrations of locally-distributed nepafenac and amfenac showed a trend of sclera > choroid > retina. Nepafenac peak levels in sub-samples posterior to the eye equator and inclusive of the posterior pole (E-PP) were 55.1, 4.03 and 2.72 nM, respectively, at 0.25 or 0.50 h, with corresponding amfenac peak levels of 41.9, 3.10 and 0.705 nM at 1 or 4 h. By comparison, peak levels in sclera, choroid and retina sub-samples in a band between the ora serrata and the equator (OS-E) were 13- to 40-fold (nepafenac) or 11- to 23-fold (amfenac) higher, indicating an anterior-to-posterior directional concentration gradient. In multiple-dosed rabbit eyes, with 0.3% nepafenac instilled once-daily or 0.1% nepafenac instilled three-times daily, cumulative 24-h locally-distributed levels of nepafenac in E-PP retina were similar between these groups, whereas exposure to amfenac once-daily dosing nepafenac 0.3% was 51% of that achieved with three-times daily dosing of 0.1%. In single-dosed monkey eyes, concentration gradients showed similar directionality as observed in rabbit eyes. Peak concentrations of locally-distributed nepafenac were 1580, 386, 292 and 13.8 nM in E-PP sclera, choroid and retina, vitreous humor, respectively, at 1 or 2 h after drug instillation. Corresponding amfenac concentrations were 21.3, 11.8, 2.58 and 2.82 nM, observed 1 or 2 h post-instillation. The data indicate that topically administered nepafenac and its metabolite amfenac reach pharmacologically relevant concentrations in the posterior eye segment (choroid and retina) via local distribution, following an anterior-to-posterior concentration gradient. The proposed pathway involves a choroidal/suprachoroidal or periocular route, along with an inward movement of drug through the sclera, choroid and retina, with negligible vitreal compartment involvement. Sustained high nepafenac concentrations in posterior segment tissues may be a reservoir for hydrolysis to amfenac.
Asunto(s)
Bencenoacetamidas/farmacocinética , Fenilacetatos/farmacocinética , Segmento Posterior del Ojo/metabolismo , Uveítis Posterior/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacocinética , Bencenoacetamidas/administración & dosificación , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Instilación de Medicamentos , Macaca fascicularis , Masculino , Soluciones Oftálmicas , Fenilacetatos/administración & dosificación , Segmento Posterior del Ojo/efectos de los fármacos , Conejos , Distribución Tisular , Uveítis Posterior/metabolismoRESUMEN
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the Western hemisphere. The recent availability of novel targeted therapies, namely Bruton's tyrosine kinase, phosphoinositide-3 kinase, and BCL-2 inhibitors, have revolutionized the treatment algorithm for CLL but have not yet resulted in cure. Advances in the field of immuno-oncology and T cell engineering brought chimeric antigen receptor (CAR) T cell therapy from the laboratory to the clinic for treatment of B cell lymphoid malignancies and has improved the disease response and survival outcomes of various types of relapsed and/or refractory B cell lymphomas. While acknowledging that there are no approved CAR T cell therapies for CLL at this time, in this comprehensive review we explore novel targets for CAR T cell therapy in CLL and highlight the promising results of CAR T cell trials reported to date. Furthermore, we shed light on future areas of development, including multitarget CAR T cell products for this disease.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfoma de Células B , Linfocitos B , Humanos , Inmunoterapia Adoptiva , Leucemia Linfocítica Crónica de Células B/terapia , Linfocitos TRESUMEN
INTRODUCTION: Long-term outcome of patients with acute myeloid leukemia (AML) remains dismal, especially for those with high-risk disease or who are refractory to conventional therapy. CAR T-cell therapy provides unique opportunity to improve outcome by specifically targeting leukemia cells through genetically engineered T cells. AREAS COVERED: We summarize the progress of CAR T-cells therapy in AML. We examine its shortcomings in AML therapy and the strategies that are being implemented to improve its safety and effectiveness. PubMed Central, ClinicalTrials.gov, and ASH annual meeting abstracts were searched. Search terms used to identify clinical trials were 'CAR T-cells in AML' OR CAR T-cells in leukemia". Relevant clinical trials and CAR T-cell research data were reviewed from June 2009 till July 2021. EXPERT OPINION: CAR T-cell therapy has shown promise as a novel therapy, but there are number of barriers to overcome to achieve its full therapeutic potential in AML. Targeting leukemia-specific antigen such as CLL1, to avoid myelotoxicity, incorporating checkpoint inhibitors to overcome leukemia-induced immunosuppression and allogenic CAR T cells to increase accessibility to patients with proliferative disease are among the strategies that are being explored to make CAR T cell a successful immunotherapy for patient with AML.
Asunto(s)
Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Quiméricos de Antígenos/uso terapéutico , Linfocitos TRESUMEN
Chimeric antigen receptors (CARs) are synthetic engineered receptors with an antigen recognition domain derived from a high-specificity monoclonal antibody that can target surface molecules on tumor cells. T cells are genetically engineered to express CARs, thereby harnessing the antigen-recognition ability of antibodies and effector function of T cells. Target surface molecule selection is crucial for manufacturing CARs. Ideally, a target surface molecule should be restricted to tumor cells and minimally expressed or absent on normal tissues. Different CD19-targeted CAR-T cell therapies have been approved for the treatment of B-cell lymphoid malignancies that are refractory to other therapies, including indolent and aggressive B-cell non-Hodgkin lymphomas (NHL) and B-cell acute lymphoblastic leukemia (B-ALL). Despite impressive results, many patients with aggressive and refractory B-cell malignancies do not respond to or relapse after CD19 CAR-T cell therapies. Thus, several additional strategies are currently being evaluated to overcome these limitations. This review discusses studies on other promising CAR-T cell targets, including CD20, CD22, BAFF-R, ROR1, CD70, BCR complex, kappa/lambda light chains, multitargeted CAR-T cells, and combinations of CAR-T cell therapy with different drugs.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Neoplasias/metabolismo , Inmunoterapia Adoptiva/métodos , Linfocitos B , Linfocitos T , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
We investigated the effects of several non-steroidal anti-inflammatory drugs on swelling related properties of mitochondria, with an emphasis on compounds that are marketed and utilized topically in the eye (nepafenac, ketorolac, diclofenac, bromfenac), and compared these to the effects of amfenac (a metabolite of nepafenac) and to celecoxib (active principle of Celebrex). With the exception of the last compound, none of the drugs promote swelling of normal mitochondria that are well energized by succinate oxidation. However, swelling is seen when the mitochondria are under an oxidative stress due to the presence of t-butylhydroperoxide. When used at 200 microM the order of potency is celecoxib > bromfenac > diclofenac > ketorolac > amfenac > nepafenac approximately equal to 0. Again with the exception of celecoxib, this swelling is not seen when mitochondria are depleted of endogenous Ca(2+) and is accelerated when exogenous Ca(2+) is provided. Sr(2+) does not substitute for exogenous Ca(2+) and prevents swelling in the presence of endogenous Ca(2+) only. The same is true for ruthenium red (inhibitor of the Ca(2+) uniporter), for cyclosporin A (inhibitor of the mitochondrial permeability transition), and for a 3.4 kDa polyethylene glycol (polymer that cancels the force which drives swelling following the permeability transition). It is concluded that several non-steroidal anti-inflammatory drugs promote the mitochondrial permeability transition under conditions of oxidative stress and in a Ca(2+) dependent fashion, whereas celecoxib functions by another mechanism. Potency of those compounds that promote the transition varies widely with bromfenac being the most potent and nepafenac having almost no effect. The mitochondrial dysfunction which is caused by the transition may underlie side effects that are produced by some of these compounds.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/química , Bencenoacetamidas/química , Bencenoacetamidas/metabolismo , Bencenoacetamidas/farmacología , Benzofenonas/química , Benzofenonas/metabolismo , Benzofenonas/farmacología , Bromobencenos/química , Bromobencenos/metabolismo , Bromobencenos/farmacología , Celecoxib , Diclofenaco/química , Diclofenaco/metabolismo , Diclofenaco/farmacología , Ketorolaco Trometamina/química , Ketorolaco Trometamina/metabolismo , Ketorolaco Trometamina/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/fisiología , Mitocondrias Hepáticas/ultraestructura , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Fenilacetatos/química , Fenilacetatos/metabolismo , Fenilacetatos/farmacología , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Temperatura , Factores de TiempoAsunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas RGS/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Encéfalo/metabolismo , Citosol/metabolismo , Cartilla de ADN , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Genoma Viral , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Isoenzimas/metabolismo , Cinética , Mamíferos , Datos de Secuencia Molecular , Fosfolipasa C beta , Reacción en Cadena de la Polimerasa/métodos , Biosíntesis de Proteínas , Subunidades de Proteína , Proteínas RGS/química , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Transducción de Señal , Transcripción Genética , Transfección/métodos , Fosfolipasas de Tipo C/metabolismoRESUMEN
The mitochondrial Ca(2+)-independent phospholipase A(2) is activated during energy-dependent Ca(2+) accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca(2+)-independent phospholipase is a factor in the mechanisms by which depolarization and Ca(2+) accumulation promote opening of the permeability transition pore. Sustained activity of the Ca(2+)-independent phospholipase A(2) promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca(2+)-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.
Asunto(s)
Citocromos c/metabolismo , Mitocondrias/enzimología , Mitocondrias/metabolismo , Fosfolipasas A/metabolismo , Animales , Apoptosis , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Medios de Cultivo/farmacología , Fosfolipasas A2 Grupo VI , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Mitocondrias Hepáticas/metabolismo , Modelos Biológicos , Necrosis , Consumo de Oxígeno , Permeabilidad , Fosfolipasas A/química , Cloruro de Potasio/química , Ratas , Factores de TiempoRESUMEN
OBJECTIVE: This study was designed to determine the role played by the mitochondrial permeability transition in the pathogenesis of mitochondrial damage and dysfunction in a representative systemic organ during the acute phase of endotoxemia. DESIGN: A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporine A, a potent inhibitor of the mitochondrial permeability transition, normalizes mitochondrial ultrastructural injury and dysfunction in the liver during acute endotoxemia. SETTING: The Ohio State University Medical Center research laboratory. SUBJECTS: Random source, adult, male conditioned cats. INTERVENTIONS: Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Treatment groups received isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 8), or cyclosporine A (6.0 mg/kg, intravenously; n = 6) or tacrolimus (FK506, 0.1 mg/kg, intravenously; n = 4) followed in 30 mins by lipopolysaccharide (3.0 mg/kg, intravenously). Liver samples were obtained 4 hrs posttreatment, and mitochondrial ultrastructure, function, and cytochrome c, Bax, and ceramide contents were assessed. MEASUREMENTS AND MAIN RESULTS: As expected, significant mitochondrial injury was apparent in the liver 4 hrs after lipopolysaccharide treatment, despite maintenance of regional tissue oxygen availability. Namely, mitochondria demonstrated high-amplitude swelling and exhibited altered respiratory function. Cyclosporine A pretreatment attenuated lipopolysaccharide-induced mitochondrial ultrastructural abnormalities and normalized mitochondrial respiratory control, reflecting protection against inner mitochondrial membrane damage. However, an abnormal permeability of outer mitochondrial membranes to cytochrome c was observed in all lipopolysaccharide-treated groups and was associated with increased mitochondrial concentrations of Bax and ceramide. CONCLUSIONS: These studies confirm that liver mitochondria are early targets of injury during endotoxemia and that inner and outer mitochondrial membrane damage occurs through different mechanisms. Inner mitochondrial membrane damage appears to relate to the mitochondrial permeability transition, whereas outer mitochondrial membrane damage can occur independent of the mitochondrial permeability transition. Preliminary evidence suggests that Bax may participate in lipopolysaccharide-induced outer mitochondrial membrane damage, but further investigations are needed to confirm this.
Asunto(s)
Endotoxemia/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Equilibrio Ácido-Base , Enfermedad Aguda , Animales , Gatos , Ciclosporina/farmacología , Membranas Intracelulares/efectos de los fármacos , Lipopolisacáridos/farmacología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Oxígeno/metabolismo , Permeabilidad/efectos de los fármacosRESUMEN
The apoptogenic protein cytochrome c can be quantitated by reverse-phase HPLC, but this method is not utilized by those who investigate mechanisms of cell death. Here, we extend the sensitivity of the method to exceed that available from immunogenic approaches and report specific procedures for applying the method to preparations of intact mitochondria, and to supernatants and pellets that arise from mitochondrial incubations. The detection limit corresponds to 0.6% of total cytochrome c found in 100 microg of rat liver mitochondrial protein, or to all of the cytochrome c that is expected in approximately 6000 hepatocytes. A single determination can be completed in 20 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedures are illustrated by experiments that determine the amount of cytochrome c released following the mitochondrial permeability transition as a function of medium ionic strength, and by long-term incubations of intact mitochondria in the presence and absence of an exogenous oxidizable substrate. Swelling and the release of adenylate kinase activity have been determined simultaneously to show how the data can be applied to evaluate the role of outer membrane disruption in mechanisms that release cytochrome c.