RESUMEN
Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that 'optimal' CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for 'real world' de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates.
Asunto(s)
Neoplasias/terapia , Receptores Quiméricos de Antígenos/aislamiento & purificación , Linfocitos T Citotóxicos/inmunología , Línea Celular Tumoral , Biblioteca de Genes , Células HEK293 , Voluntarios Sanos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Neoplasias/patología , Cultivo Primario de Células , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/trasplanteRESUMEN
BACKGROUND: At the time of upfront treatment failure, over half of the patients with advanced pancreatic cancer are candidates for further treatment. METHODS: Patients with metastatic gemcitabine-resistant pancreatic cancer were treated with mitomycin 8 mg/m(2) on day 1, ifosfamide 2,500 mg/m(2) and mesna 3,000 mg/m(2) on days 1-3 every 28 days until progressive disease. A positive responder was defined as a patient who was progression free at 6 months from trial enrollment. According to the Fleming design, a sample size of 34 patients was estimated assuming p0 = 0.05 and p1 = 0.20. RESULTS: Between May 2006 and December 2007, 21 patients (median age 56 years; median Karnofsky performance score 80) were enrolled. One patient died before receiving any treatment. Eighteen patients interrupted chemotherapy due to progressive disease (n = 15), toxicity (n = 2) or refusal (n = 1). Grade >2 toxicity consisted of neutropenia in 80% of patients, thrombocytopenia and fatigue in 20% and anemia in 10%. Only 1 patient was progression free at 6 months (5%). One patient had a partial response (5%) and 2 patients had stable disease (10%). Median survival was 3.7 months. CONCLUSION: Based on the poor outcome observed and on the high level of grade 3-4 toxicity, the trial was prematurely stopped and the mitomycin and ifosfamide regimen was considered insufficiently active in gemcitabine-pretreated advanced pancreatic cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Esquema de Medicación , Resistencia a Antineoplásicos , Femenino , Humanos , Ifosfamida/administración & dosificación , Ifosfamida/efectos adversos , Masculino , Persona de Mediana Edad , Mitomicina/administración & dosificación , Mitomicina/efectos adversos , Terapia Recuperativa/métodos , GemcitabinaRESUMEN
Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2nd generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1+ tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Tumoral , Células Clonales , Femenino , Humanos , Ratones , Unión Proteica , Multimerización de Proteína , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos de Cadena Única/inmunología , Solubilidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.
Asunto(s)
Anticuerpos/análisis , Técnicas de Visualización de Superficie Celular/métodos , Secuencia de Aminoácidos , Antígenos/metabolismo , Línea Celular , Epítopos/metabolismo , Escherichia coli/metabolismo , Humanos , Inmunoterapia , Dominios Proteicos , Anticuerpos de Cadena Única/inmunología , Linfocitos T/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/ß-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated ß-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Regeneración/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Péptidos/metabolismo , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genética , Pez CebraRESUMEN
BACKGROUND: More than half of patients with pancreatic adenocarcinoma (PA) are candidates for further treatment when they experience upfront treatment failure. PATIENTS AND METHODS: Patients with gemcitabine-resistant PA, age <76 years and Karnofski performance status (KPS) >50 were treated with a XELIRI or FOLFIRI regimen until progressive disease or a maximum of six months. As this was an observational study, no statistical design was performed. RESULTS: Between July 2007 and December 2009, 34 patients (median age 60 years; median KPS 90) were treated with XELIRI (26) or FOLFIRI (8) regimen. Grade >2 toxicity consisted of neutropenia in 9% of patients, anemia and fatigue in 3% and hand-foot syndrome in 12%. Median progression-free survival was two months (range 1-4). Maximum response was stable disease in four patients (12%). Median survival was 4.2 (range 1-15) months. CONCLUSION: Fluoropyrimidine and irinotecan combination does not seem to have any role in the treatment of gemcitabine-resistant PA.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Terapia Recuperativa , Adenocarcinoma/secundario , Adolescente , Adulto , Anciano , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Capecitabina , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/análogos & derivados , Humanos , Irinotecán , Leucovorina/administración & dosificación , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Tasa de Supervivencia , Resultado del Tratamiento , Adulto Joven , GemcitabinaRESUMEN
Core-binding factor leukemia (CBFL) is a subgroup of acute myeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most common mutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3' untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133(+) stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.
Asunto(s)
Subunidades alfa del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide/genética , MicroARNs/genética , Antígeno AC133 , Enfermedad Aguda , Adolescente , Adulto , Anciano , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Regulación hacia Abajo , Eritropoyetina/farmacología , Femenino , Citometría de Flujo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Mutación , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937RESUMEN
Human tryptase is a serine protease expressed in mast-cells. We previously observed that AML blast cells, cultured in vitro from a KIT D816Y patient, give rise to adherent cells with mast-cell like phenotype and tryptase was released in the serum-free medium. To correlate total serum tryptase (ts-try) levels with cytogenetic features and KIT mutational status, we analyzed serum samples from AML patients at diagnosis. In 70 out of 155 patients (45%) we detected elevated ts-try (>15 ng/mL), significantly linked to t(8;21) (P < .001) and inv(16) (P = .007). In patients that achieved complete remission the ts-try decreased to normal values. In 75 patients screened for KIT mutation, we found a clear relationship between elevated ts-try and mutated patients with t(8;21) (P < .001). In conclusion, we propose that checking for ts-try at diagnosis of AML may be a simple tool to select patients to be addressed to KIT mutation screening.