RESUMEN
BACKGROUND: Human epidermal growth factor receptor 3 (HER3) is broadly expressed in non-small-cell lung cancer (NSCLC) and is the target of patritumab deruxtecan (HER3-DXd), an antibody-drug conjugate consisting of a HER3 antibody attached to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. U31402-A-U102 is an ongoing phase I study of HER3-DXd in patients with advanced NSCLC. Patients with epidermal growth factor receptor (EGFR)-mutated NSCLC that progressed after EGFR tyrosine kinase inhibitor (TKI) and platinum-based chemotherapy (PBC) who received HER3-DXd 5.6 mg/kg intravenously once every 3 weeks had a confirmed objective response rate (cORR) of 39%. We present median overall survival (OS) with extended follow-up in a larger population of patients with EGFR-mutated NSCLC and an exploratory analysis in those with acquired genomic alterations potentially associated with resistance to HER3-DXd. PATIENTS AND METHODS: Safety was assessed in patients with EGFR-mutated NSCLC previously treated with EGFR TKI who received HER3-DXd 5.6 mg/kg; efficacy was assessed in those who also had prior PBC. RESULTS: In the safety population (N = 102), median treatment duration was 5.5 (range 0.7-27.5) months. Grade ≥3 adverse events occurred in 76.5% of patients; the overall safety profile was consistent with previous reports. In 78/102 patients who had prior third-generation EGFR TKI and PBC, cORR by blinded independent central review (as per RECIST v1.1) was 41.0% [95% confidence interval (CI) 30.0% to 52.7%], median progression-free survival was 6.4 (95% CI 4.4-10.8) months, and median OS was 16.2 (95% CI 11.2-21.9) months. Patients had diverse mechanisms of EGFR TKI resistance at baseline. At tumor progression, acquired mutations in ERBB3 and TOP1 that might confer resistance to HER3-DXd were identified. CONCLUSIONS: In patients with EGFR-mutated NSCLC after EGFR TKI and PBC, HER3-DXd treatment was associated with a clinically meaningful OS. The tumor biomarker characterization comprised the first description of potential mechanisms of resistance to HER3-DXd therapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Carcinoma de Pulmón de Células no Pequeñas , Receptores ErbB , Neoplasias Pulmonares , Mutación , Receptor ErbB-3 , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Receptores ErbB/genética , Receptores ErbB/antagonistas & inhibidores , Femenino , Receptor ErbB-3/genética , Receptor ErbB-3/antagonistas & inhibidores , Persona de Mediana Edad , Masculino , Anciano , Adulto , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Anciano de 80 o más Años , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Camptotecina/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos ampliamente neutralizantes , Inmunoconjugados/uso terapéutico , Inmunoconjugados/efectos adversos , Inmunoconjugados/administración & dosificaciónRESUMEN
BACKGROUND: Patritumab deruxtecan (HER3-DXd) is a human epidermal growth factor receptor 3 (HER3)-directed antibody-drug conjugate composed of a fully human anti-HER3 monoclonal antibody (patritumab) covalently linked to a topoisomerase I inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. TOT-HER3 is a window-of-opportunity study designed to assess the biological activity, measured by CelTIL score [= -0.8 × tumor cellularity (in %) + 1.3 × tumor-infiltrating lymphocytes (TILs) (in %)], and clinical activity of HER3-DXd during short-term (21 days) pre-operative treatment in patients with primary operable HER2-negative early breast cancer. PATIENTS AND METHODS: Patients with previously untreated hormone receptor-positive/HER2-negative tumors were allocated to one of four cohorts according to baseline ERBB3 messenger RNA expression. All patients received one dose of HER3-DXd 6.4 mg/kg. The primary objective was to evaluate change from baseline in CelTIL score. RESULTS: Seventy-seven patients were evaluated for efficacy. A significant change in CelTIL score was observed, with a median increase from baseline of 3.5 (interquartile range, -3.8 to 12.7; P = 0.003). Among patients assessable for clinical response (n = 62), an overall response rate of 45% was observed (tumor measurement by caliper), with a trend toward an increase in CelTIL score among responders compared with non-responders (mean difference, +11.9 versus +1.9). Change in CelTIL score was independent of baseline ERBB3 messenger RNA and HER3 protein levels. Genomic changes occurred, including switching toward a less proliferative tumor phenotype based on PAM50 subtypes, suppression of cell proliferation genes, and induction of genes associated with immunity. Treatment-emergent adverse events were observed in 96% of patients (14% grade ≥3); most common were nausea, fatigue, alopecia, diarrhea, vomiting, abdominal pain, and neutrophil count decrease. CONCLUSIONS: A single dose of HER3-DXd was associated with clinical response, increased immune infiltration, suppression of proliferation in hormone receptor-positive/HER2-negative early breast cancer, and a tolerable safety profile consistent with previously reported results. These findings support further study of HER3-DXd in early breast cancer.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Receptor ErbB-2/metabolismo , Camptotecina/uso terapéutico , Trastuzumab/uso terapéuticoRESUMEN
Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Receptores de Citocinas/fisiología , Receptores Inmunológicos/fisiología , Transducción de Señal/fisiología , Trombopoyetina/metabolismo , Transactivadores/metabolismo , División Celular , Células Cultivadas , Activación Enzimática , Humanos , Mutagénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Receptores de Trombopoyetina , TYK2 QuinasaRESUMEN
Recent studies have suggested that members of the Abl interactor (Abi) protein family negatively regulate cell growth and transformation. To date, however, no specific role in these cellular processes has been identified for the Abi family. Here we describe the inhibition by overexpressed Abi-1 of a mitogenic pathway activated by both growth factors and v-Abl. We have identified the guanine nucleotide exchange factors Sos1 and Sos2 as novel binding partners of Abi-1. A domain that is required for interaction with Sos in vivo has been mapped to the amino terminus of Abi-1. Overexpression of Abi-1 inhibits epidermal growth factor (EGF)-induced activation of extracellular signal-regulated kinases (Erks) but does not affect EGF-induced activation of c-Jun N-terminal kinase or Akt. In addition, overexpression of Abi-1 blocks Erk activation induced by v-Abl. In both cases, the maximal inhibitory effect requires an intact amino-terminal Sos-binding domain in Abi-1. Finally, we demonstrate that tyrosine phosphorylation of endogenous Abi-1 in fibroblasts is induced by both v-Abl and serum stimulation, further suggesting a role for Abi-1 in signal transduction initiated by v-Abl and growth factors. Taken together, these findings suggest that overexpressed Abi proteins negatively regulate cell growth and transformation by specifically targeting the Erk pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Oncogénicas v-abl/antagonistas & inhibidores , Proteína SOS1/metabolismo , Proteínas Son Of Sevenless/metabolismo , Animales , Línea Celular , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Proteína Adaptadora GRB2 , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Oncogénicas v-abl/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Dominios Homologos src/genéticaRESUMEN
Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.
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Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Animales , Anticuerpos , Fraccionamiento Celular , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/inmunología , Fibroblastos/metabolismo , Ratones , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/inmunología , Fosforilación , Pruebas de Precipitina , Procesamiento Proteico-Postraduccional , Factor de Respuesta SéricaRESUMEN
Adenoassociated virus (AAV) has been developed as a vector for gene transfer because of its advantageous features: it is nonpathogenic, naturally replication-defective; it infects growth-arrested cells, and can transfer the therapeutic gene without co-delivery of any viral genes. However, a major obstacle in conducting systematic studies of AAV-mediated gene transfer in animal models is the difficulty of obtaining large quantities of recombinant virus. Recent development of AAV packaging cell lines has simplified the procedure of producing recombinant AAV (rAAV). However, the efficacy of producing large quantities of rAAV with these cell lines is yet to be demonstrated. In this study we have analyzed the difference between the replication of wild-type AAV and the production of rAAV. Using a combined single-plasmid system that carries both an AAV vector and the rep-cap genes, we have demonstrated that the AAV vector replicates to high number of copies whereas the rep-cap sequences remain unamplified in the virus-producing cells, When the copy number of rep-cap genes was increased by varying the vector/rep-cap ratio in the transfection mixture, the titer of rAAV increased proportionally. Thus, the titer of rAAV is limited by the low copy number of the rep-cap genes that results in an insufficient expression of the Rep and Cap proteins. We have also shown that generation of double-stranded replicating form of the vector DNA is accompanied by an amplified transgene expression. We propose that the increased gene expression from the accumulating double-stranded viral DNA is likely to be the mechanism by which wild-type AAV produces a large number of particles necessary to package the self-replicating AAV genomes. We conclude that mimicking this amplified expression of rep-cap genes may provide the key to produce high titers of rAAV.
Asunto(s)
Replicación del ADN/genética , Dependovirus/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Southern Blotting , ADN Recombinante/genética , ADN Recombinante/metabolismo , Dosificación de Gen , Regulación de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Genes Reporteros , Genes Virales/genética , Vectores Genéticos/genética , Células HeLa , Virus Helper/genética , Virus Helper/metabolismo , Histocitoquímica , Humanos , Plásmidos/genética , Transfección/genéticaRESUMEN
Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively-increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a "head-full" mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging.
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ADN Viral/química , Dependovirus/genética , Dependovirus/fisiología , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Genoma Viral , Ensamble de Virus/genética , Southern Blotting , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , ADN Viral/análisis , Técnicas de Transferencia de Gen , Genes Reporteros , Células HeLa , Humanos , Recombinación Genética , TransfecciónRESUMEN
O6-Alkylguanine-DNA-alkyltransferase (O6-AGT) is a very important DNA repair protein known to carry out the transfer of alkyl groups from the O6 position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. In this work, the activity of O6-AGT in different cell lines and the relationship between the depletion of the enzyme and the frequency of micronuclei induced by cisplatin (DDP), Ning Xin platin (camphoramine chloroacetic platinum, CCP) or carboplatin (JM-8) in KB and CHL cells were studied. Experiments indicate that KB cells showed higher O6-AGT activity (greater than 400 dpm/300 micrograms protein extracts) which belonged to Mer+ cells, but CHL, HL-60 and L1210 cells showed very low O6-AGT activity (less than 50 dpm/300 micrograms protein extracts) which can be considered to be Mer- cells. Cytotoxicity studies indicated that no mer- selection was observed in these platinum complexes for KB, HL-60, CHL and L1210 cells. However, a good relationship between the depletion of O6-AGT and the frequency of micronuclei induced by the platinum complexes was obtained. CCP caused the highest depletion of the enzyme and exhibited highest potency in damaging chromosome.
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Carboplatino/toxicidad , Cromosomas/efectos de los fármacos , Cisplatino/toxicidad , Metiltransferasas/metabolismo , Compuestos Organoplatinos/toxicidad , Animales , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Células KB/efectos de los fármacos , Células KB/enzimología , Leucemia L1210/genética , Leucemia L1210/patología , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Pruebas de Micronúcleos , O(6)-Metilguanina-ADN Metiltransferasa , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
TPO is a recently cloned cytokine which appears to play a central role in megakaryopoiesis and platelet production. It has been shown to be the ligand for c-mpl, a member of the hematopoietic receptor superfamily. We examined intracellular tyrosine phosphorylation events induced by TPO and compared them with events stimulated by IL-3, a pleiotropic cytokine which also has activity on the megakaryocyte lineage. The overall pattern of tyrosine phosphorylation stimulated by TPO and IL-3 in myeloid precursor cells revealed an overlapping but not identical pattern reflecting their distinct but partially redundant biological effects. We identify Shc and Vav as intracellular targets of TPO-induced tyrosine phosphorylation. Moreover, we demonstrate that the tyrosine kinase Jak2 is phosphorylated after TPO stimulation. Whereas phosphorylation of these proteins was induced by both cytokines, phosphorylation of Jak1 was induced only by IL-3 and not by TPO, distinguishing the signal transduction of the two cytokines on a molecular level.
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Proteínas de Ciclo Celular , Interleucina-3/farmacología , Proteínas de Neoplasias , Proteínas Tirosina Quinasas/biosíntesis , Receptores Inmunológicos/metabolismo , Trombopoyetina/metabolismo , Tirosina/análogos & derivados , Animales , Línea Celular , Inducción Enzimática , Células Madre Hematopoyéticas , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Ratones , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-vav , Receptores de Citocinas/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores de Trombopoyetina , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Trombopoyetina/biosíntesis , Trombopoyetina/aislamiento & purificación , Transfección , Tirosina/aislamiento & purificación , Tirosina/metabolismoRESUMEN
Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.