RESUMEN
SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive IHC studies, which corroborated published RNA sequencing data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer. SGN-CD228A was efficiently internalized in various tumor cell types, and its cytotoxic activity was dependent on CD228 expression and internalization and intrinsic sensitivity to the MMAE payload. Compared with the valine-citrulline dipeptide linker, the novel glucuronide linker increased the cellular retention of MMAE in vitro and conferred improved antitumor activity against melanoma cell lines in vitro and in vivo. In addition, SGN-CD228A was active across melanoma, TNBC, and NSCLC cell line- and patient-derived xenograft models with heterogeneous antigen expression. In vivo, CD228 expression was important for response to SGN-CD228A but was not well correlated across all tumor types, suggesting that other factors associated with ADC activity are important. Overall, SGN-CD228A is a CD228-directed, investigational ADC that employs innovative technology and has compelling preclinical antitumor activity. SGN-CD228A is investigated in a Phase I clinical trial (NCT04042480) in patients with advanced solid tumors.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Inmunoconjugados , Neoplasias Pulmonares , Melanoma , Neoplasias de la Mama Triple Negativas , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Glucurónidos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inmunoconjugados/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000-280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (< or = 20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoconjugados/uso terapéutico , Melanoma/tratamiento farmacológico , Proteínas de Neoplasias/inmunología , Oligopéptidos/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Biomarcadores de Tumor/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Humanos , Melanoma/inmunología , Antígenos Específicos del Melanoma , Ratones , Neoplasias Cutáneas/inmunología , Células Tumorales CultivadasRESUMEN
Experiments in vascular smooth muscle cells (SMCs) indicate that the transcription factor cAMP response element-binding protein (CREB), the cyclic nucleotide response element-binding protein, suppresses expression of the platelet-derived growth factor-alpha receptor gene (PDGFRalpha). Adenovirus-mediated expression of constitutively active CREB mutants decreases PDGFRalpha mRNA, PDGFRalpha protein, and PDGFRalpha promoter-luciferase reporter activity in cultured SMCs. Expression of dominant negative CREB protein, A-CREB, increases PDGFRalpha protein content and the PDGFRalpha-promoter activity in SMCs. Active CREB prevents activation of PDGFRalpha promoter-luciferase reporter activity by CCAAT/enhancer-binding protein-delta (C/EBPdelta), shown to mediate IL-1beta stimulation of PDGFRalpha expression. Exposure of cultured SMCs to high glucose or reactive oxidant stress, which decrease CREB protein content and activity, increases PDGFRalpha protein content and promoter activity. Expression of active CREB blunts reactive oxidant stress-induced PDGFRalpha accumulation in SMCs. Loss of CREB protein in aortic walls of rats with streptozotocin-induced diabetes is accompanied by an increase in PDGFRalpha content. In Ob/Ob mice (which demonstrate reduced aortic wall CREB content vs. Ob/- controls), treatment with the peroxisomal proliferator-activated receptor gamma rosiglitazone increases CREB content and decreases PDGFRalpha content in the aortic wall. Thus, both in vitro and in vivo loss of CREB content and activity and subsequent accumulation of PDGFRalpha may contribute to SMC activation during diabetes.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Músculo Liso Vascular/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/análisis , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/análisis , Diabetes Mellitus Experimental/metabolismo , Resistencia a la Insulina , Músculo Liso Vascular/citología , Estrés Oxidativo , ARN Mensajero/análisis , Ratas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
In this article, we describe a novel antibody-drug conjugate (ADC; SGN-LIV1A), targeting the zinc transporter LIV-1 (SLC39A6) for the treatment of metastatic breast cancer. LIV-1 was previously known to be expressed by estrogen receptor-positive breast cancers. In this study, we show that LIV-1 expression is maintained after hormonal therapy in primary and metastatic sites and is also upregulated in triple-negative breast cancers. In addition to breast cancer, other indications showing LIV-1 expression include melanoma, prostate, ovarian, and uterine cancer. SGN-LIV1A consists of a humanized antibody conjugated through a proteolytically cleavable linker to monomethyl auristatin E, a potent microtubule-disrupting agent. When bound to surface-expressed LIV-1 on immortalized cell lines, this ADC is internalized and traffics to the lysozome. SGN-LIV1A displays specific in vitro cytotoxic activity against LIV-1-expressing cancer cells. In vitro results are recapitulated in vivo where antitumor activity is demonstrated in tumor models of breast and cervical cancer lineages. These results support the clinical evaluation of SGN-LIV1A as a novel therapeutic agent for patients with LIV-1-expressing cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Transporte de Catión/antagonistas & inhibidores , Inmunoconjugados/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Afinidad de Anticuerpos , Antineoplásicos/administración & dosificación , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Inmunoconjugados/administración & dosificación , Inmunofenotipificación , Lisosomas/metabolismo , Células MCF-7 , Microtúbulos/metabolismo , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR(+), HEL9217 and TF1-alpha, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNFalpha-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR(-) and MDR(+) AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.
Asunto(s)
Anticuerpos Monoclonales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células HL-60 , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Ratones SCID , Fagocitosis , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Resultado del Tratamiento , Células U937RESUMEN
Cytokines are known to induce apoptosis of pancreatic beta-cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic beta-cell line, MIN6 cells, to cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced beta-cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of beta-cells.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Islotes Pancreáticos/fisiología , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Genes Reporteros , Islotes Pancreáticos/efectos de los fármacos , Luciferasas/genética , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.