RESUMEN
Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
Asunto(s)
Anticuerpos/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoconjugados/farmacología , Inmunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animales , Anticuerpos/genética , Ligando CD27/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoconjugados/química , Maitansina/metabolismo , Ratones , Ratones SCID , Receptores Fc/genéticaRESUMEN
Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Inmunoconjugados/metabolismo , Maitansina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Antineoplásicos Fitogénicos/administración & dosificación , Línea Celular Tumoral , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/administración & dosificación , Lisosomas/metabolismo , Maitansina/administración & dosificaciónRESUMEN
Antibody drug conjugates are emerging as a powerful class of antitumor agents with efficacy across a range of cancers; therefore, understanding the disposition of this class of therapeutic is crucial. Reported here is a method of enriching a specific organelle (lysosome) to understand the catabolism of an anti-CD70 Ab-MCC-DM1, an antibody drug conjugate with a noncleavable linker. With such techniques a higher degree of concentration-activity relationship can be established for in vitro cell lines; this can aid in understanding the resultant catabolite concentrations necessary to exert activity.
Asunto(s)
Inmunoconjugados/metabolismo , Lisosomas/metabolismo , Preparaciones Farmacéuticas/metabolismo , Ligando CD27/inmunología , Línea Celular Tumoral , HumanosRESUMEN
Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential mediator of osteoclast formation, function and survival. In patients with solid tumor metastasis to the bone, targeting the bone microenvironment by inhibition of RANKL using denosumab, a fully human monoclonal antibody (mAb) specific to RANKL, has been demonstrated to prevent tumor-induced osteolysis and subsequent skeletal complications. Recently, a prominent functional role for the RANKL pathway has emerged in the primary bone tumor giant cell tumor of bone (GCTB). Expression of both RANKL and RANK is extremely high in GCTB tumors and denosumab treatment was associated with tumor regression and reduced tumor-associated bone lysis in GCTB patients. In order to address the potential role of the RANKL pathway in another primary bone tumor, this study assessed human RANKL and RANK expression in human primary osteosarcoma (OS) using specific mAbs, validated and optimized for immunohistochemistry (IHC) or flow cytometry. Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79) of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy would not directly affect the tumor.
RESUMEN
INTRODUCTION: Bone metastasis is a serious complication in patients with lung cancer, occurring in up to 40% of patients. Tumor cell-mediated osteolysis occurs ultimately through induction of RANK ligand (RANKL) within the bone stroma although this hypothesis has not been tested extensively in the setting of non-small-cell lung cancer (NSCLC). By using two novel NSCLC bone metastasis mouse models, we examined the effects of RANKL inhibition on osteolysis and tumor progression. METHODS: We treated mice bearing skeletal NSCLC tumors with osteoprotegerin-Fc (OPG-Fc) to assess whether osteoclast inhibition through RANKL inhibition would affect bone metastases at early or late stages of bone colonization. Progression of skeletal tumor was determined by radiography, longitudinal bioluminescent imaging, and histological analyses. RESULTS: OPG-Fc reduced development and progression of radiographically evident osteolytic lesions and also significantly reduced skeletal tumor progression in both NSCLC bone metastasis models. In the H1299 human NSCLC bone metastasis model, OPG-Fc plus docetaxel in combination resulted in significantly greater inhibition of skeletal tumor growth compared with either single agent alone. The observed ability of RANKL inhibition to reduce NSCLC osteolytic bone destruction or skeletal tumor burden was associated with decreases in tumor-associated osteoclasts. CONCLUSIONS: These results demonstrate that RANKL is required for the development of tumor-induced osteolytic bone destruction caused by NSCLC cells in vivo. RANKL inhibition also reduced skeletal tumor burden, presumably through the indirect mechanism of blocking tumor-induced osteoclastogenesis and resultant production of growth factors and calcium from the bone microenvironment. RANKL inhibition also provided an additive benefit to docetaxel treatment by augmenting the reduction of tumor burden.
Asunto(s)
Neoplasias Óseas/prevención & control , Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Enoxaparina/farmacología , Neoplasias Pulmonares/prevención & control , Osteólisis/tratamiento farmacológico , Osteoprotegerina/metabolismo , Ligando RANK/antagonistas & inhibidores , Animales , Anticoagulantes/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Osteoprotegerina/inmunología , Tasa de Supervivencia , Carga Tumoral , Células Tumorales CultivadasRESUMEN
RANK ligand (RANKL), acting through its cognate receptor RANK, is a key factor for bone remodeling and metastasis by regulating the differentiation, survival and activation of osteoclasts. RANKL is also crucial for the development of mouse mammary glands during pregnancy and has been recently linked to the etiology of breast cancer via its direct activity on RANK-expressing normal or transformed breast epithelial cells, leading to increased mitogenesis, enhanced regenerative potential of mammary stem cells, and increased invasion and migration. We demonstrate that higher RANK expression in MDA-MB-231 breast cancer cells (MDA-231-RANK cells) is sufficient to confer a significantly greater metastatic growth rate in the bone compared with MDA-MB-231 cells which do not express high levels of RANK. Blockade of osteoclastic bone resorption, achieved with treatment by either RANKL inhibition or zoledronic acid, did reduce skeletal tumor progression of MDA-231-RANK cells suggesting that the vicious cycle contributes to metastatic growth. However, RANKL inhibition reduced skeletal growth of MDA-231-RANK tumors to a significantly greater extent than zoledronic acid, indicating that skeletal growth of RANK-positive tumors is also driven by direct RANKL effects. RANKL stimulated the expression of multiple genes associated with cell invasive behavior, including several matrix metalloproteinases and other genes previously defined as part of a bone metastasis gene signature. These data indicate that RANKL provokes breast cancer bone metastases via two distinct, but potentially overlapping mechanisms: stimulation of tumor-associated osteoclastogenesis and stimulation of RANK-expressing tumor cells.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Ligando RANK/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , HumanosRESUMEN
Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial-mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44(+)CD24(-) cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis.
Asunto(s)
Neoplasias de la Mama/etiología , Transformación Celular Neoplásica , Transición Epitelial-Mesenquimal , Receptor Activador del Factor Nuclear kappa-B/fisiología , Animales , Proteína BRCA1/fisiología , Neoplasias de la Mama/patología , Antígeno CD24/análisis , Línea Celular Tumoral , Movimiento Celular , Humanos , Receptores de Hialuranos/análisis , Ratones , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Ligando RANK/análisis , Receptor Activador del Factor Nuclear kappa-B/análisisRESUMEN
Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.
Asunto(s)
Diferenciación Celular/fisiología , Osteoclastos/citología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/fisiología , Fosfatasa Ácida/genética , Animales , Proteínas Portadoras/farmacología , Células Cultivadas , Sinergismo Farmacológico , Embrión de Mamíferos , Inhibidores Enzimáticos , Femenino , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes src/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Quinasa I-kappa B , Proteínas I-kappa B/metabolismo , Queratinocitos/citología , Hígado , Factor Estimulante de Colonias de Macrófagos/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Noqueados , Inhibidor NF-kappaB alfa , FN-kappa B/fisiología , Osteoclastos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Quinasa de Factor Nuclear kappa BRESUMEN
Signaling through receptor activator of nuclear factor-kappaB (RANK) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate RANK ligand are profoundly osteopetrotic because of the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular tumor necrosis factor receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cDNA into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the interleukin-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts.