RESUMO
Patients with relapsed/refractory B-cell malignancies such as non-Hodgkin lymphoma (B-NHL) or acute lymphoblastic leukemia have a poor prognosis. Despite measurable clinical activity with new targeted therapies, many patients do not achieve a complete or durable response suggesting an opportunity to improve upon existing therapies. Here we describe SGN-CD19B, a pyrrolobenzodiazepine (PBD)-based anti-CD19 antibody drug conjugate (ADC) being investigated for treatment of B-cell malignancies, which has improved potency compared with other ADCs. CD19-expressing tumor cells rapidly internalize SGN-CD19B, and the released PBD drug induces DNA damage, resulting in G2/M cell cycle arrest and cell death. SGN-CD19B demonstrated activity against a broad panel of malignant B-cell lines and induced durable regressions in mice bearing xenografts derived from these B-cell malignancies. A single dose of SGN-CD19B induced durable regressions at 300 µg/kg (3 µg/kg drug equivalents); combination with rituximab decreased the curative dose to 100 µg/kg (1 µg/kg drug equivalents). These doses are significantly lower than the level of drug required with other ADC payloads. In cynomolgus monkeys, SGN-CD19B effectively depleted CD20+ B lymphocytes in peripheral blood and lymphoid tissues confirming that SGN-CD19B is pharmacodynamically active at well-tolerated doses. In summary, preclinical studies show SGN-CD19B is a highly active ADC, which releases a DNA cross-linking agent rather than a microtubule inhibitor. The distinct mechanism of action, broad potency, and potential to combine with rituximab suggest that SGN-CD19B may offer unique clinical opportunities in B-cell malignancies. A phase 1 clinical trial is in progress to investigate the therapeutic potential of SGN-CD19B in relapsed/refractory B-NHL. This trial was registered at www.clinicaltrials.gov as #NCT02702141.
Assuntos
Anticorpos/uso terapêutico , Antígenos CD19/metabolismo , Benzodiazepinas/química , Linfoma de Células B/tratamento farmacológico , Pirróis/química , Animais , Anticorpos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Linfoma de Células B/patologia , Macaca fascicularis , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.
Assuntos
Anticorpos Monoclonais Humanizados/química , Benzodiazepinas/química , Resistencia a Medicamentos Antineoplásicos , Imunoconjugados/química , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/química , Animais , Apoptose , Ciclo Celular , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/genética , Dimerização , Desenho de Fármacos , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/imunologia , CamundongosRESUMO
Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR.
Assuntos
Imunoconjugados/farmacologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Humanos , Imunoconjugados/imunologia , Leucemia Mieloide Aguda/imunologia , Camundongos , Camundongos SCID , Células THP-1 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SOST, a novel bone morphogenetic protein (BMP) antagonist and negative regulator of bone formation, is expressed in osteogenic cells. Null mutations in the SOST gene are associated with the sclerosteosis phenotype typified by high bone mass. We sought to delineate the pathways involved in the regulation of SOST expression in human osteoblastic cells. We evaluated the effects of bone growth factors and hormones on the RNA levels of SOST and the BMP antagonists, noggin and gremlin. Parathyroid hormone (PTH), transforming growth factor-beta1 (TGF-beta1), fibroblast growth factors 1 and 2 (FGF1, FGF2), and insulin-like growth factor-1 (IGF-1) had negligible effects on SOST expression in human osteoblasts. In comparison, BMPs-2, 4, and 6 induced the message levels of SOST in a time- and dose-dependent manner. The levels of noggin and, to a lesser extent, gremlin were also increased by BMPs. BMP's stimulatory effects on SOST were further enhanced by retinoic acid or 1,25-dihydroxyvitamin D3. In contrast, dexamethasone (DEX) blocked the effects of the BMPs on SOST and gremlin, but not on noggin. Retinoic acid and 1,25-dihydroxyvitamin D3 did not affect the BMP-enhanced expression of gremlin or noggin. The steroids did not affect the endogenous levels of the BMP antagonists. These findings show that the levels of SOST are modulated by BMPs and the interactions of the BMPs with steroid hormones in human osteoblasts. These effects differed markedly from that of noggin or gremlin, suggesting that there is an exquisite regulation of the expressions of BMP antagonists in cells of the osteoblast lineage.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Calcitriol/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/fisiologia , Marcadores Genéticos/genética , Osteoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A null mutation in the SOST gene is associated with sclerosteosis, an inherited disorder characterized by a high bone mass phenotype. The protein product of the SOST gene, sclerostin, is a bone morphogenetic protein (BMP) antagonist that decreases osteoblast activity and reduces the differentiation of osteoprogenitors. We sought to delineate the mechanism by which sclerostin modulated osteoblastic function by examining the effects of the protein on differentiating cultures of human mesenchymal stem cells (hMSC). Sclerostin significantly decreased alkaline phosphatase (ALP) activity and the proliferation of hMSC cells. In addition, hMSC cells treated with sclerostin displayed a marked increase in caspase activity. Elevated levels of fragmented histone-associated DNA in these cells were detected by ELISA and by TUNEL staining. Other BMP antagonists including noggin, Chordin, Gremlin, and Twisted gastrulation did not affect caspase activity. The sclerostin-mediated increase in caspase activity was blocked by caspase-1 and caspase-3 inhibitors. Sclerostin-induced changes in ALP activity and the survival of hMSC cells were partially restored by BMP-6, suggesting the involvement of additional growth factors. These findings show that sclerostin selectively controls the apoptosis of bone cells. The ability of sclerostin to interact with important growth factors such as BMPs likely serves as the basis by which it modulates the survival of osteoblasts. By making these growth factors unavailable for cell function, sclerostin promotes the apoptosis of bone cells, providing a novel level of control in the regulation of bone formation.
Assuntos
Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Inibidores de Caspase , Caspases/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Marcadores Genéticos/genética , Humanos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genéticaRESUMO
Despite therapeutic advances, the poor prognoses for acute myeloid leukemia (AML) and intermediate and high-risk myelodysplastic syndromes (MDS) point to the need for better treatment options. AML and MDS cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, induced meaningful responses in a Phase 1 clinical trial and demonstrated anti-leukemic activity in preclinical models. Recently, it was reported that 5-azacytidine (Vidaza™) prolonged the overall survival of a group of high risk MDS and AML patients. To determine whether the combination of lintuzumab and 5-azacytidine would be beneficial, a mouse xenograft model of disseminated AML was used to evaluate the combination. There was a significant reduction in tumor burden and an increase in overall survival in mice treated with lintuzumab and 5-azacytidine. The effects were greater than that obtained with either agent alone. As the in vivo anti-leukemic activity of lintuzumab was dependent upon the presence of mouse effector cells including macrophages and neutrophils, in vitro effector function assays were used to assess the impact of 5-azacytidine on lintuzumab activity. The results show that 5-azacytidine significantly enhanced the ability of lintuzumab to promote tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytic (ADCP) activities. These results suggest that lintuzumab and 5-azacytidine act in concert to promote tumor cell killing. Additionally, these findings provide the rationale to evaluate this combination in the clinic.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Azacitidina/administração & dosagem , Sinergismo Farmacológico , Leucemia Mieloide Aguda/terapia , Macrófagos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Células HL-60 , Humanos , Leucemia Mieloide Aguda/imunologia , Macrófagos/imunologia , Camundongos , Camundongos SCID , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Anticorpos Monoclonais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Antineoplásicos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Células HL-60 , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos SCID , Fagocitose , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Resultado do Tratamento , Células U937RESUMO
The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.
Assuntos
Antígeno Ki-1/química , Lisossomos/metabolismo , Oligopeptídeos/química , Anticorpos/química , Antígenos CD20/química , Antineoplásicos/farmacologia , Western Blotting , Catepsina B/química , Linhagem Celular , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Endocitose , Endopeptidases/química , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Microscopia de Fluorescência , Modelos Químicos , Peptídeo Hidrolases/química , Peptídeos/química , Ligação Proteica , Frações Subcelulares/metabolismo , Temperatura , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 6 , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Marcadores Genéticos , Glicoproteínas , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C3H , Modelos Biológicos , Mutação , Osteoblastos/metabolismo , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta CateninaRESUMO
Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum-free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed alpha-fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4alpha. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast-like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy-1), CD34, and OV-6 but do not stain with antibodies to beta(2)-microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.
Assuntos
Técnicas Citológicas , Hepatócitos/citologia , Hepatócitos/fisiologia , Fígado/embriologia , Biomarcadores/análise , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Criopreservação , Feto/citologia , Inibidores do Crescimento/farmacologia , Hepatócitos/metabolismo , Humanos , Oncostatina M , Organoides/citologia , Peptídeos/farmacologia , Fatores de TempoRESUMO
Noggin and sclerostin are bone morphogenetic protein (BMP) antagonists that modulate mitogenic activity through sequestering BMPs. Little is known of the interactions among this class of proteins. We show that recombinant sclerostin and noggin bound to each other with high affinity (K(D) = 2.92 nm). This observation has been extended to naturally expressed noggin and sclerostin from the rat osteosarcoma cell line, ROS 17/2.8, supporting a role for the complex in natural systems. The noggin-sclerostin complex was competitive with BMP binding and mutually attenuated the activity of each BMP antagonist. Collectively, the data demonstrate a novel and exquisite paradigm for the regulation of BMP activity through direct neutralization of the BMP and activation by co-localized BMP antagonist expression. The pleiotrophic nature of noggin and sclerostin represents a novel mechanism for the fine-tuning of BMP activity in bone homeostasis.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Ligação Competitiva , Western Blotting , Proteína Morfogenética Óssea 6 , Osso e Ossos/metabolismo , Proteínas de Transporte , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Glicoproteínas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Camundongos , Camundongos Endogâmicos C3H , Osteossarcoma/metabolismo , Testes de Precipitina , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Smad , Fatores de Tempo , Transativadores/metabolismoRESUMO
There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.