RESUMEN
Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2- and VEGF-mediated phosphorylation of ERK1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases (PTP) by oxidizing an essential cysteine residue in the active site. ATN-224-mediated inhibition of SOD1 in tumor and endothelial cells prevents the formation of sufficiently high levels of H(2)O(2), resulting in the protection of PTPs from H(2)O(2)-mediated oxidation. This, in turn, leads to the inhibition of EGF-, IGF-1-, and FGF-2-mediated phosphorylation of ERK1/2. Pretreatment with exogenous H(2)O(2) or with the phosphatase inhibitor vanadate abrogates the inhibition of ERK1/2 phosphorylation induced by ATN-224 or SOD1 siRNA treatments. Furthermore, ATN-224-mediated SOD1 inhibition causes the down-regulation of the PDGF receptor. SOD1 inhibition also increases the steady-state levels of superoxide, which induces protein oxidation in A431 cells but, surprisingly, does not oxidize phosphatases. Thus, SOD1 inhibition in A431 tumor cells results in both prooxidant effects caused by the increase in the levels of superoxide and antioxidant effects caused by lowering the levels of H(2)O(2). These results identify SOD1 as a master regulator of GF signaling and as a therapeutic target for the inhibition of angiogenesis and tumor growth.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Oxígeno/química , Monoéster Fosfórico Hidrolasas/metabolismo , Superóxido Dismutasa/fisiología , Línea Celular Tumoral , Endotelio Vascular/citología , Humanos , Modelos Biológicos , Molibdeno/farmacología , Neovascularización Patológica , Oxidación-Reducción , Fosforilación , Especies Reactivas de Oxígeno , Transducción de Señal , Superóxido Dismutasa-1RESUMEN
PURPOSE: ATN-161 (Ac-PHSCN-NH(2)) is an integrin-binding peptide that is currently in phase II trials in cancer patients. This peptide has been shown to have antitumor activity in a number of different preclinical models. EXPERIMENTAL DESIGN: In this study, we examined the binding, biodistribution, and dose and biomarker response of ATN-161 in several animal models. RESULTS: ATN-161 bound to the beta subunit of a number of different integrins implicated in tumor growth and progression, which depended on its cysteine thiol. The peptide had antiangiogenic activity in the Matrigel plug model, and this activity could be reversed by inhibitors of protein kinase A, an effector of alpha(5)beta(1)-dependent angiogenesis. A labeled analogue of ATN-161, ATN-453, localized to neovessels but not to preexisting vasculature in vivo. The half-life of the peptide when localized to a tumor was much longer than in plasma. Dose-response studies in the Matrigel plug model of angiogenesis or a Lewis lung carcinoma model of tumor growth showed a U-shaped dose-response curve with 1 to 10 mg/kg given thrice a week, being the optimal dose range of ATN-161. Two additional pharmacodynamic models of angiogenesis (dynamic contrast-enhanced magnetic resonance imaging and measurement of endothelial cell progenitors) also revealed U-shaped dose-response curves. CONCLUSIONS: The presence of a U-shaped dose-response curve presents a significant challenge to identifying a biologically active dose of ATN-161. However, the identification of biomarkers of angiogenesis that also exhibit this same U-shaped response should allow the translation of those biomarkers to the clinic, allowing them to be used to identify the active dose of ATN-161 in phase II studies.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Oligopéptidos/farmacología , Animales , Biomarcadores de Tumor/análisis , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
PURPOSE: Copper chelation reduces the secretion of many angiogenic factors and reduces tumor growth and microvascular density in animal models. ATN-224 is a second-generation analogue of ammonium tetrathiomolybdate. The aim of our phase I study was to reduce serum copper levels, as measured by ceruloplasmin, to 5 to 15 mg/dL (normal 16-60) in 14 to 21 days, to determine the pharmacokinetic profile of ATN-224 and to evaluate dose-limiting toxicities. PATIENTS AND METHODS: Cohorts of patients were treated with escalating oral doses of ATN-224 until copper depletion followed by a titrated maintenance dose. RESULTS: Eighteen patients received 78 cycles of ATN-224. Mean baseline ceruloplasmin was 39.6 mg/dL. The maximum administered dose was 330 mg/d where grade 3 fatigue was dose-limiting. At the maximum tolerated dose of 300 mg/d, the median time to achieve target ceruloplasmin was 21 days, and toxicities included grade 3 anemia, grade 3 neutropenia, fatigue, and sulfur eructation. ATN-224 treatment caused a significant reduction (> 90%) in RBC superoxide dismutase 1 activity and circulating endothelial cells. Pharmacokinetic data indicate greater absorption of ATN-224 and more rapid ceruloplasmin reduction when administered with a proton pump inhibitor. Stable disease of > 6 months was observed in 2 patients. CONCLUSIONS: Oral ATN-224 is a well-tolerated therapy and at a loading dose of 300 mg/d leads to a reduction of serum ceruloplasmin levels in 80% patients within 21 days. A loading dose of 300 mg/d for 2 weeks followed by a titrated maintenance dose will be the recommended starting dose for phase II study.
Asunto(s)
Quelantes/efectos adversos , Quelantes/farmacocinética , Quelantes/uso terapéutico , Terapia por Quelación , Colina/efectos adversos , Colina/farmacocinética , Colina/uso terapéutico , Cobre/sangre , Molibdeno/efectos adversos , Molibdeno/farmacocinética , Molibdeno/uso terapéutico , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Ceruloplasmina/efectos de los fármacos , Citocinas/sangre , Citocinas/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Superóxido Dismutasa/sangre , Superóxido Dismutasa/efectos de los fármacosRESUMEN
Antibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSAKIT D816V and ROSAKIT D816V-Gluc), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F). In these cell lines, AGS-16C3F induced cell apoptosis at very low concentrations. To characterize the effects of AGS-16C3F on leukemia progression in vivo, ROSAKIT D816V-Gluc NOD-SCID γ mouse models of advanced SM (AdvSM) were treated with AGS-16C3F or an ADC control for 2 weeks. Whereas AGS-16C3F had no apparent toxicity in xenotransplanted mice, in vivo neoplastic MC burden significantly decreased in both hematopoietic and nonhematopoietic organs. Furthermore, animals treated with AGS-16C3F had prolonged survival compared with the animals treated with control ADC, and AGS-16C3F efficiently prevented disease relapse. In conclusion, these preclinical studies identified CD203c as a novel therapeutic target on neoplastic MCs, and AGS-16C3F as a promising ADC for the treatment of patients with AdvSM.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Inmunoconjugados/uso terapéutico , Mastocitosis Sistémica/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Mastocitosis Sistémica/patología , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Purpose: To determine the safety, pharmacokinetics, and recommended phase II dose of an antibody-drug conjugate (ADC) targeting ectonucleotide phosphodiesterases-pyrophosphatase 3 (ENPP3) conjugated to monomethyl auristatin F (MMAF) in subjects with advanced metastatic renal cell carcinoma (mRCC).Patients and Methods: Two phase I studies were conducted sequentially with 2 ADCs considered equivalent, hybridoma-derived AGS-16M8F and Chinese hamster ovary-derived AGS-16C3F. AGS-16M8F was administered intravenously every 3 weeks at 5 dose levels ranging from 0.6 to 4.8 mg/kg until unacceptable toxicity or progression. The study was terminated before reaching the MTD. A second study with AGS-16C3F started with the AGS-16M8F bridging dose of 4.8 mg/kg given every 3 weeks.Results: The AGS-16M8F study (n = 26) closed before reaching the MTD. The median duration of treatment was 12 weeks (1.7-83 weeks). One subject had durable partial response (PR; 83 weeks) and 1 subject had prolonged stable disease (48 weeks). In the AGS-16C3F study (n = 34), the protocol-defined MTD was 3.6 mg/kg, but this was not tolerated in multiple doses. Reversible keratopathy was dose limiting and required multiple dose deescalations. The 1.8 mg/kg dose was determined to be safe and was associated with clinically relevant signs of antitumor response. Three of 13 subjects at 1.8 mg/kg had durable PRs (range, 100-143 weeks). Eight subjects at 2.7 mg/kg and 1.8 mg/kg had disease control >37 weeks (37.5-141 weeks).Conclusions: AGS-16C3F was tolerated and had durable antitumor activity at 1.8 mg/kg every 3 weeks. Clin Cancer Res; 24(18); 4399-406. ©2018 AACR.
Asunto(s)
Anticuerpos Antiidiotipos/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Inmunoconjugados/administración & dosificación , Oligopéptidos/administración & dosificación , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genética , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antiidiotipos/efectos adversos , Células CHO , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Cricetulus , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoconjugados/efectos adversos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Metástasis de la Neoplasia , Oligopéptidos/efectos adversos , Hidrolasas Diéster Fosfóricas/inmunología , Pirofosfatasas/inmunologíaRESUMEN
AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Epitelio Corneal/efectos de los fármacos , Inmunoconjugados/toxicidad , Pinocitosis/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Macaca fascicularis , Masculino , Modelos Animales , Conejos , Homología de Secuencia de AminoácidoRESUMEN
PURPOSE: A second-generation tetrathiomolybdate analogue (ATN-224; choline tetrathiomolybdate), which selectively binds copper with high affinity, is currently completing two phase I clinical trials in patients with advanced solid and advanced hematologic malignancies. However, there is very little information about the mechanism of action of ATN-224 at the molecular level. EXPERIMENTAL DESIGN: The effects of ATN-224 on endothelial and tumor cell growth were evaluated in cell culture experiments in vitro. The antiangiogenic activity of ATN-224 was investigated using the Matrigel plug model of angiogenesis. RESULTS: ATN-224 inhibits superoxide dismutase 1 (SOD1) in tumor and endothelial cells. The inhibition of SOD1 leads to inhibition of endothelial cell proliferation in vitro and attenuation of angiogenesis in vivo. The inhibition of SOD1 activity in endothelial cells is dose and time dependent and leads to an increase in the steady-state levels of superoxide anions, resulting in the inhibition of extracellular signal-regulated kinase phosphorylation without apparent induction of apoptosis. In contrast, the inhibition of SOD1 in tumor cells leads to the induction of apoptosis. The effects of ATN-224 on endothelial and tumor cells could be substantially reversed using Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, a catalytic small-molecule SOD mimetic. CONCLUSIONS: These data provide a distinct molecular target for the activity of ATN-224 and provide validation for SOD1 as a target for the inhibition of angiogenesis and tumor growth.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Cobre/metabolismo , Molibdeno/farmacología , Mieloma Múltiple/tratamiento farmacológico , Superóxido Dismutasa/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mieloma Múltiple/irrigación sanguínea , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Fosforilación/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
PURPOSE: Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to alpha5beta1 and alphavbeta3 in vitro, to block breast cancer growth and metastasis. EXPERIMENTAL DESIGN: MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05-1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. RESULT: Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. CONCLUSION: These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Neoplasias de la Mama/tratamiento farmacológico , Oligopéptidos/farmacología , Adenocarcinoma/patología , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Radiografía/instrumentación , Neoplasias de los Tejidos Blandos/prevención & control , Neoplasias de los Tejidos Blandos/secundario , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877-86. ©2017 AACRSee related article by Zhao et al., p. 1866.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Inmunoconjugados/farmacología , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Pinocitosis , Antineoplásicos Inmunológicos/efectos adversos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoconjugados/efectos adversos , Megacariocitos/metabolismo , Activación Plaquetaria/efectos de los fármacos , Transporte de Proteínas , Receptores de IgG/metabolismo , Trombocitopenia/sangre , Trombocitopenia/inducido químicamenteRESUMEN
Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866-76. ©2017 AACRSee related article by Zhao et al., p. 1877.
Asunto(s)
Antineoplásicos/efectos adversos , Inmunoconjugados/efectos adversos , Mielopoyesis/efectos de los fármacos , Neutropenia/sangre , Neutropenia/etiología , Neutrófilos/efectos de los fármacos , Animales , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Ratones , Neutrófilos/metabolismo , Pinocitosis , Receptores de IgG/metabolismo , Serina Proteasas/metabolismoRESUMEN
A series of novel, multisubstrate, bicyclic pyrimidine nucleoside inhibitors of human thymidine phosphorylase (TP) is described. Thymidine phosphorylase has been implicated in angiogenesis and plays a significant role in tumor progression and metastasis. The presence and orientation of the phosphonate moiety (acting as a phosphate mimic) in these derivatives were critical for inhibitory activity. The most active compounds possessed a phosphonate group in an endo orientation. This was consistent with molecular modeling results that showed the endo isomer protein-ligand complex to be lower in energy than the exo complex.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Inhibidores Enzimáticos/síntesis química , Nucleósidos de Pirimidina/síntesis química , Timidina Fosforilasa/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Relación Estructura-ActividadRESUMEN
High molecular weight kininogen (HK) is an abundant, multi-domain plasma protein that circulates in plasma primarily in its single chain form. Proteolytic cleavage of HK by plasma kallikrein releases the vasoactive nanopeptide bradykinin (BK), and converts HK into two-chain HK (HKa). BK appears to have pro-angiogenic activity, most likely mediated through binding to B1 and B2 receptors on endothelial cells. Conversely, HKa and its domain 5, but not (single chain) HK, have potent anti-angiogenic activity comparable to other endogenous angiogenesis inhibitors. The mechanism by which HKa exerts its anti-angiogenic activity remains controversial, but appears to involve binding to cell surface tropomyosin and induction of apoptosis of proliferating endothelial cells. A role for tropomyosin in mediating the anti-angiogenic signals of other anti-angiogenic proteins such as endostatin and histidine-proline-rich glycoprotein (HPRG) has also been reported. Here we review the physiological importance of high molecular weight kininogen in angiogenesis, with emphasis on the mechanism(s) by which this activity is mediated.
Asunto(s)
Quininógeno de Alto Peso Molecular/farmacología , Neovascularización Patológica/prevención & control , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Humanos , Quininógeno de Alto Peso Molecular/química , Quininógeno de Alto Peso Molecular/metabolismo , Datos de Secuencia Molecular , Calicreína Plasmática/metabolismoRESUMEN
Prostate-specific membrane antigen (PSMA), a glutamyl preferring carboxypeptidase, is found in prostate and other carcinomas present on both tumor cells and associated microvascular lining cells. We find that the channel structures delineated by PSMA-expressing cells in human and rat prostate tumors are in functional continuity with the vasculature and thus form part of tumor microvasculature. The PSMA-positive cell-outlined channels are CD31 negative and mutually exclusive of CD31-positive cell-lined channels elsewhere in the tumor consistent with tumor cells adapted to a pseudoendothelial phenotype in vasculogenic mimicry. To assess the functional potential of such PSMA-lined microvasculature to selectively direct infarctive tumor therapy, we coupled the soluble extracellular domain of tissue factor to a PSMA catalytic site inhibitor to create a PSMA-directed selective tumor vascular thrombogen (STVT). This protein induced selective local in vivo infarctive necrosis of the rat Mat Lu prostate tumor when administered i.v. The combined administration of this STVT with low-dose doxorubicin produced a profound tumoricidal effect, resulting in complete eradication of some tumors. This is consistent with the therapeutic potential for a PSMA-directed STVT and expands the potential for selective infarctive ablation of tumors.
Asunto(s)
Antígenos de Superficie , Carboxipeptidasas/antagonistas & inhibidores , Dipéptidos/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Sitios de Unión , Carboxipeptidasas/biosíntesis , Catálisis , Dipéptidos/administración & dosificación , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Glutamato Carboxipeptidasa II , Humanos , Infarto/inducido químicamente , Liposomas , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Ratas , Tromboplastina/farmacología , Trombosis/inducido químicamente , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline-rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in fibroblast growth factor-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor growth in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 approximately 600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a 16-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor growth, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Antineoplásicos/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Tropomiosina/metabolismo , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Pollos , Endotelio Vascular/efectos de los fármacos , Humanos , Cinética , Melanoma Experimental/tratamiento farmacológico , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/farmacología , RatasRESUMEN
Histidine-proline-rich glycoprotein (HPRG) is an abundant multidomain plasma protein evolutionarily related to high-molecular-weight kininogen. The cleaved form of high-molecular-weight kininogen has recently been demonstrated to exhibit antiangiogenic activities in vitro (J. C. Zhang et al., FASEB J., 14: 2589-2600, 2000), mediated primarily through domain 5. HPRG contains a histidine-proline-rich (H/P) domain with sequence and functional similarities to HKa-D5. We hypothesized that HPRG may also have antiangiogenic properties, localized within its H/P domain. The H/P domain is highly conserved among species, and because rabbit H/P domain is more resistant to internal proteolytic cleavage than the human domain, the rabbit HPRG (rbHPRG) was primarily used to assess the antiangiogenic activity of HPRG. Rabbit HPRG inhibited human umbilical vein endothelial cell (HUVEC) tube formation stimulated by fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor on a Matrigel surface as well as cell proliferation of FGF-2 stimulated HUVECs. The antiangiogenic activity of rbHPRG was localized to the H/P domain by use of proteolytic fragments of rbHPRG and was further confirmed and characterized in two in vivo models of angiogenesis: the chorioallantoic membrane of the chick assay and the mouse Matrigel plug assay. Caspase-3 activation was observed in HUVECs stimulated with FGF-2 in the presence of rbHPRG, suggesting that apoptosis of activated endothelial cells may be one of the mechanisms underlying its antiangiogenic activity. Finally, the H/P domain of rbHPRG reduced tumor cell number when tumor cells were co-inoculated in the Matrigel plug assay. In conclusion, the H/P domain within HPRG induces the apoptosis of activated endothelial cells leading to potent antiangiogenic effects.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Proteínas/farmacología , Inhibidores de la Angiogénesis/fisiología , Animales , División Celular/efectos de los fármacos , Embrión de Pollo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Plasminógeno/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Proteínas/fisiología , Conejos , Secuencias Repetitivas de Aminoácido , Homología de Secuencia de AminoácidoRESUMEN
SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoconjugados/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Oligopéptidos/química , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Neoplasias Renales/metabolismo , Ratones , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Regulación hacia Arriba , Urotelio/metabolismo , Urotelio/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Inmunoconjugados/farmacología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Pirofosfatasas/antagonistas & inhibidores , Animales , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Renales/tratamiento farmacológico , Macaca fascicularis , Ratones , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Oligopéptidos/inmunología , Animales , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Macaca fascicularis , Ratones , Ratones Endogámicos ICR , Ratones SCID , Nectinas , Neoplasias/enzimología , Neoplasias/patología , Ratas , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma/metabolismo , Inmunoconjugados/farmacología , Proteínas de Transporte de Membrana/metabolismo , Oligopéptidos/farmacología , Neoplasias Pancreáticas/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Carcinoma/tratamiento farmacológico , Carcinoma/genética , Carcinoma/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Angiogenesis is characterized by the development of new vasculature from pre-existing vessels and plays a central role in physiological processes such as embryogenesis, wound healing and female reproductive function, as well as pathophysiologic events including cancer, rheumatoid arthritis and diabetic retinopathy. The growth and metastasis of tumors is critically dependent upon angiogenesis. Although targeting angiogenesis as a therapeutic strategy has to date met with limited success in the clinic, the recent FDA approval of the anti-VEGF antibody Avastin has validated the use of anti-angiogenic therapeutic strategies for cancer treatment. We have recently identified several plasma proteins having anti-angiogenic properties, including Histidine-Proline-Rich Glycoprotein (HPRG) and activated high-molecular-weight kininogen (HKa). Both of these proteins are able to induce apoptosis in endothelial cells in vitro and can inhibit angiogenesis in vivo. Recent studies from our laboratories have also identified a novel cell-surface binding protein for HKa that mediates its anti-angiogenic activity. This protein, tropomyosin, is normally found inside the cell and is associated with the actin cytoskeleton, where it plays a critical role in stabilizing actin filaments in a variety of cell types. However, in angiogenic endothelial cells, tropomyosin appears to have extracellular localization. Previous studies have also suggested the involvement of tropomyosin in the anti-angiogenic activity of endostatin, and our recent work indicates that tropomyosin may mediate the antiangiogenic activity of HPRG as well. In this review, we summarize data describing extracellular tropomyosin as a novel receptor for multiple anti-angiogenic proteins. Extracellular tropomyosin may therefore represent a previously undescribed central target for the development of anti-angiogenic therapy.