RESUMEN
The low-density lipoprotein receptor (LDLR) is a membrane receptor that mediates the endocytosis of low-density lipoprotein (LDL). Uptake of LDL has been proposed to contribute to chemotherapy resistance of acute myeloid leukaemia (AML) cell lines in vitro. In the present study, we analysed LDLR expression and survival using bone marrow biopsies from 187 intensively treated patients with AML. Here, increasing LDLR expression was associated with decreasing overall (58·4%, 44·2%, and 24·4%; P = 0·0018), as well as event-free survival (41·7%, 18·1%, and 14·3%; P = 0·0077), and an increasing cumulative incidence of relapse (33·9%, 55·1%, and 71·4%; P = 0·0011). Associations of LDLR expression with survival were confirmed in 557 intensively treated patients from two international validation cohorts. In the analytic and validation cohorts, LDLR expression remained associated with outcome in multivariable regression analyses including the European LeukemiaNet genetic risk classification. Thus, LDLR predicts outcome of patients with AML beyond existing risk factors. Furthermore, we found low expression levels of LDLR in most healthy tissues, suggesting it as a promising target for antibody-based pharmacodelivery approaches in AML.
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Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Receptores de LDL/genética , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/metabolismo , Médula Ósea/patología , Femenino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Pronóstico , Receptores de LDL/análisis , Adulto JovenRESUMEN
Capsaicin (CAP) has been long known for its analgesic properties and more recently for its antitumor activity in various cell types. However, its pungency and the high doses needed to achieve a significant activity have precluded its application in cancer therapy. Herein, we propose a straightforward novel strategy to improve the antitumor effect of CAP based on the enhancement of its aggregation propensity in aqueous media by covalent attachment of a BODIPY (BDP) dye. The target CAP-BDP 1 self-assembles in aqueous solutions into weakly fluorescent globular assemblies that become highly emissive upon cell uptake-induced disassembly. Remarkably, due to the improved delivery to the tumour tissue upon aggregation, we have succeeded in reducing the doses of CAP-based drugs in vivo in prostate cancer by two orders of magnitude while maintaining a substantial antitumor activity.
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Antineoplásicos/farmacología , Compuestos de Boro/química , Capsaicina/farmacología , Colorantes Fluorescentes/química , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Antineoplásicos/química , Compuestos de Boro/síntesis química , Capsaicina/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Colorantes Fluorescentes/síntesis química , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Células PC-3 , Neoplasias de la Próstata/patologíaRESUMEN
tTF-NGR retargets the extracellular domain of tissue factor via a C-terminal peptide GNGRAHA, a ligand of the surface protein aminopeptidase N (CD13) and upon deamidation of integrin αvß3, to tumor vasculature. tTF-NGR induces tumor vascular infarction with consecutive antitumor activity against xenografts and selectively inhibits tumor blood flow in cancer patients. Since random PEGylation resulted in favorable pharmacodynamics of tTF-NGR, we performed site-directed PEGylation of PEG units to the N-terminus of tTF-NGR to further improve the antitumor profile of the molecule. Mono-PEGylation to the N-terminus did not change the procoagulatory activity of the tTF-NGR molecule as measured by Factor X activation. Experiments to characterize pharmacokinetics in mice showed a more than 1 log step higher mean area under the curve of PEG20k-tTF-NGR over tTF-NGR. Acute (24 h) tolerability upon intravenous application for the mono-PEGylated versus non-PEGylated tTF-NGR compounds was comparable. PEG20k-tTF-NGR showed clear antitumor efficacy in vivo against human tumor xenografts when systemically applied. However, site-directed mono-PEGylation to the N-terminus does not unequivocally improve the therapeutic profile of tTF-NGR.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Tromboplastina/uso terapéutico , Animales , Línea Celular Tumoral , Clonación Molecular , Humanos , Espectrometría de Masas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/irrigación sanguínea , Polietilenglicoles/metabolismo , Dominios y Motivos de Interacción de Proteínas , Tromboplastina/químicaRESUMEN
OBJECTIVES: To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. METHODS: For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). RESULTS: Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. CONCLUSIONS: Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy.
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Inhibidores de la Angiogénesis/farmacología , Fibrosarcoma/terapia , Infarto/etiología , Tromboplastina/farmacología , Terapia por Ultrasonido , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Endotelio Vascular , Femenino , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/tratamiento farmacológico , Citometría de Flujo , Humanos , Ratones , Ratones Desnudos , Microburbujas , Neovascularización Patológica/prevención & controlRESUMEN
The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.
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Inhibidores de la Angiogénesis/farmacología , Infarto , Angiografía por Resonancia Magnética , Neoplasias Experimentales , Tromboplastina/farmacología , Tomografía Computarizada de Emisión de Fotón Único , Animales , Línea Celular Tumoral , Humanos , Infarto/inducido químicamente , Infarto/diagnóstico por imagen , Ratones , Ratones Desnudos , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Radiografía , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Tromboplastina/genéticaRESUMEN
Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.
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Crisis Blástica/metabolismo , Crisis Blástica/mortalidad , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Osteopontina/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Estudios de Cohortes , Supervivencia sin Enfermedad , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Tasa de SupervivenciaRESUMEN
ABSTRACT: Antibody-based immunotherapies have revolutionized leukemia and lymphoma treatment, with animal studies being crucial in evaluating effectiveness and side effects. By targeting the evolutionary conserved Slamf7 immune receptor, which is naturally expressed by the murine multiple myeloma cell line MPC-11, we have developed a syngeneic mouse model for direct comparison of 3 immunotherapies: monoclonal antibodies (mAb), bispecific T-cell engagers (BiTE), and chimeric antigen receptor (CAR) T cells (CART), all targeting Slamf7. Slamf7-BiTE is a bispecific single-chain antibody consisting of α-Slamf7 and α-CD3 Fv fragments joined through a Gly-Ser linker, and Slamf7-CART comprises the α-Slamf7 Fv fragment fused to the msCD8α transmembrane and msCD28, 4-1BB, and CD3ζ intracellular signaling domains. Slamf7-BiTE and Slamf7-CART effectively killed MPC-11 cells in vitro, independently of Slamf7-mediated inhibitory signaling by self-ligation. After chimerizing the constant region of the rat-anti-mouse Slamf7 antibody to mouse Fc-immunoglobulin G2a for enhanced effector functions, Slamf7-mAb triggered antigen-specific antibody-dependent cellular cytotoxicity by binding to Fcγ receptor IV. In vivo, all 3 immunotherapies showed antitumor effects against Slamf7-expressing targets. Unlike Slamf7-mAb, Slamf7-BiTE led to considerable side effects in test animals, including weight loss and general malaise, which were also observed to a lesser extent after Slamf7-CART infusion. In allogeneic transplant, Slamf7-BiTE and Slamf7-CART maintained activity compared with the nontransplant setting, whereas Slamf7-mAb displayed enhanced antimyeloma activity. In summary, our model faithfully replicates treatment efficacy and side effects detected after human immunotherapy. It aids in developing and improving immunotherapies and may help devise novel approaches to mitigate undesired effects in steady state and allogeneic stem cell transplantation.
Asunto(s)
Anticuerpos Biespecíficos , Mieloma Múltiple , Ratones , Ratas , Humanos , Animales , Mieloma Múltiple/tratamiento farmacológico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia , Anticuerpos Biespecíficos/uso terapéuticoRESUMEN
To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Linfocitos T , Células Endoteliales , Línea Celular Tumoral , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Muerte Celular , Hipoxia/metabolismo , Inmunoterapia Adoptiva , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/terapia , Neoplasias/metabolismoRESUMEN
Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx(m2) ). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx(m2) with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx(m2) -mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxC¹¹³S exchange in Trx(m2) . Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cisteína/metabolismo , Citosol/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clonación Molecular , Genes Reporteros , Prueba de Complementación Genética , Glucosafosfato Deshidrogenasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Mutación , Cebollas/genética , Cebollas/metabolismo , Peroxisomas/metabolismo , Filogenia , Plastidios/genética , Plastidios/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Nanomedicines have enormous potential to improve the precision of cancer therapy, yet our ability to efficiently home these materials to regions of disease in vivo remains very limited. Inspired by the ability of communication to improve targeting in biological systems, such as inflammatory-cell recruitment to sites of disease, we construct systems where synthetic biological and nanotechnological components communicate to amplify disease targeting in vivo. These systems are composed of 'signalling' modules (nanoparticles or engineered proteins) that target tumours and then locally activate the coagulation cascade to broadcast tumour location to clot-targeted 'receiving' nanoparticles in circulation that carry a diagnostic or therapeutic cargo, thereby amplifying their delivery. We show that communicating nanoparticle systems can be composed of multiple types of signalling and receiving modules, can transmit information through multiple molecular pathways in coagulation, can operate autonomously and can target over 40 times higher doses of chemotherapeutics to tumours than non-communicating controls.
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Nanomedicina/métodos , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Coagulación Sanguínea , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Humanos , Ratones , Modelos Biológicos , Trasplante de Neoplasias , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Péptidos/química , Transducción de Señal , TemperaturaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target "undruggable" oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML. METHODS: Our AML-targeting system consists of an internalizing anti-CD33-antibody-protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application. RESULTS: The nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton's kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation. CONCLUSIONS: We report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML.
Asunto(s)
Leucemia Mieloide Aguda , Protaminas , Humanos , Ratones , Animales , Electricidad Estática , ARN Interferente Pequeño/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Metiltransferasas , ADNRESUMEN
We induced thrombosis of blood vessels in solid tumors in mice by a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA, targeting aminopeptidase N (CD13) and the integrin alpha(v)beta(3) (CD51/CD61) on tumor vascular endothelium. The designed fusion protein tTF-NGR retained its thrombogenic activity as demonstrated by coagulation assays. In vivo studies in mice bearing established human adenocarcinoma (A549), melanoma (M21), and fibrosarcoma (HT1080) revealed that systemic administration of tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels as shown by histologic analysis. tTF-NGR, but not untargeted tTF, induced significant tumor growth retardation or regression in all 3 types of solid tumors. Thrombosis induction in tumor vessels by tTF-NGR was also shown by contrast enhanced magnetic resonance imaging (MRI). In the human fibrosarcoma xenograft model, MRI revealed a significant reduction of tumor perfusion by administration of tTF-NGR. Clinical first-in-man application of low dosages of this targeted coagulation factor revealed good tolerability and decreased tumor perfusion as measured by MRI. Targeted thrombosis in the tumor vasculature induced by tTF-NGR may be a promising strategy for the treatment of cancer.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Tromboplastina/antagonistas & inhibidores , Adulto , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Células Cultivadas , Sistemas de Liberación de Medicamentos/métodos , Embolia/inducido químicamente , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Neoplasias/irrigación sanguínea , Oligopéptidos/metabolismo , Terapia Recuperativa , Tromboplastina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Besides its central functional role in coagulation, TF has been described as being operational in the development of malignancies and is currently being studied as a possible therapeutic tool against cancer. One of the avenues being explored is retargeting TF or its truncated extracellular part (tTF) to the tumor vasculature to induce tumor vessel occlusion and tumor infarction. To this end, multiple structures on tumor vascular wall cells have been studied at which tTF has been aimed via antibodies, derivatives, or as bifunctional fusion protein through targeting peptides. Among these targets were vascular adhesion molecules, oncofetal variants of fibronectin, prostate-specific membrane antigens, vascular endothelial growth factor receptors and co-receptors, integrins, fibroblast activation proteins, NG2 proteoglycan, microthrombus-associated fibrin-fibronectin, and aminopeptidase N. Targeting was also attempted toward cellular membranes within an acidic milieu or toward necrotic tumor areas. tTF-NGR, targeting tTF primarily at aminopeptidase N on angiogenic endothelial cells, was the first drug candidate from this emerging class of coaguligands translated to clinical studies in cancer patients. Upon completion of a phase I study, tTF-NGR entered randomized studies in oncology to test the therapeutic impact of this novel therapeutic modality.
RESUMEN
Early assessment of target hit in anti-cancer therapies is a major task in oncologic imaging. In this study, immediate target hit and effectiveness of CD13-targeted tissue factor tTF-NGR in patients with advanced malignant disease enrolled in a phase I trial was assessed using a multiparametric MRI protocol. Seventeen patients with advanced solid malignancies were enrolled in the trial and received tTF-NGR for at least one cycle of five daily infusions. Tumor target lesions were imaged with multiparametric MRI before therapy initiation, five hours after the first infusion and after five days. The imaging protocol comprised ADC, calculated from DWI, and DCE imaging and vascular volume fraction (VVF) assessment. DCE and VVF values decreased within 5 h after therapy initiation, indicating early target hit with a subsequent decrease in tumor perfusion due to selective tumor vessel occlusion and thrombosis induced by tTF-NGR. Simultaneously, ADC values increased at five hours after tTF-NGR administration. In four patients, treatment had to be stopped due to an increase in troponin T hs, with subsequent anticoagulation. In these patients, a reversed effect, with DCE and VVF values increasing and ADC values decreasing, was observed after anticoagulation. Changes in imaging parameters were independent of the mean vessel density determined by immunohistochemistry. By using a multiparametric imaging approach, changes in tumor perfusion after initiation of a tumor vessel occluding therapy can be evaluated as early as five hours after therapy initiation, enabling early assessment of target hit.
RESUMEN
Targeted therapies against cancer have become more and more important. In particular, the inhibition of tumor angiogenesis and vascular targeting have been the focus of new treatment strategies. Numerous new substances were developed as angiogenesis inhibitors and evaluated in clinical trials for safety, tolerance, and efficacy. With positive study results, some of these molecules have already been approved for clinical use. For example, this is true for the vascular endothelial growth factor neutralizing antibody bevacizumab (BEV) in metastatic colorectal cancer, nonsmall cell lung cancer, renal cancer, and breast cancer. The tyrosine kinase (TK) inhibitors sorafenib and sunitinib have been approved for metastatic renal cancer as well as for hepatocellular carcinoma, and sunitinib has also been approved for gastrointestinal stroma tumors. In this chapter we try to give an overview of the substances currently investigated in Phase III studies and beyond with regard to antiangiogenesis in cancer therapy.
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Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias/tratamiento farmacológico , Axitinib , Bencenosulfonatos/uso terapéutico , Ensayos Clínicos Fase III como Asunto , Endostatinas/uso terapéutico , Humanos , Imidazoles/uso terapéutico , Indazoles/uso terapéutico , Indoles/uso terapéutico , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/uso terapéutico , Pirroles/uso terapéutico , Sorafenib , Sunitinib , Talidomida/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
BACKGROUND: CD-13 targeted tissue factor tTF-NGR is a fusion protein selectively inducing occlusion of tumor vasculature with resulting tumor infarction. Mechanistic and pharmacodynamic studies have shown broad anti-tumor therapeutic effects in xenograft models. METHODS: After successful Good Manufacturing Practice (GMP) production and before translation into clinical phase I, ICH S9 (S6) guideline-conforming animal safety, toxicology, and pharmacokinetic (PK) studies were requested by the federal drug authority in accordance with European and US regulations. RESULTS: These studies were performed in mice, rats, guinea pigs, and beagle dogs. Results of the recently completed clinical phase I trial in end-stage cancer patients showed only limited predictive value of these non-clinical studies for patient tolerability and safety in phase I. CONCLUSIONS: Although this experience cannot be generalized, alternative pathways with seamless clinical phase 0 microdosing-phase I dose escalation studies are endorsed for anticancer drug development and translation into the clinic.
RESUMEN
BACKGROUND: Truncated tissue factor (tTF) retargeted by NGR-peptides to aminopeptidase N (CD13) in tumor vasculature is effective in experimental tumor therapy. tTF-NGR induces tumor growth inhibition in a variety of human tumor xenografts of different histology. To improve on the therapeutic efficacy we have combined tTF-NGR with radiotherapy. METHODS: Serum-stimulated human umbilical vein endothelial cells (HUVEC) and human HT1080 sarcoma cells were irradiated in vitro, and upregulated early-apoptotic phosphatidylserine (PS) on the cell surface was measured by standard flow cytometry. Increase of cellular procoagulant function in relation to irradiation and PS cell surface concentration was measured in a tTF-NGR-dependent Factor X activation assay. In vivo experiments with CD-1 athymic mice bearing human HT1080 sarcoma xenotransplants were performed to test the systemic therapeutic effects of tTF-NGR on tumor growth alone or in combination with regional tumor ionizing radiotherapy. RESULTS: As shown by flow cytometry with HUVEC and HT1080 sarcoma cells in vitro, irradiation with 4 and 6 Gy in the process of apoptosis induced upregulation of PS presence on the outer surface of both cell types. Proapoptotic HUVEC and HT1080 cells both showed significantly higher procoagulant efficacy on the basis of equimolar concentrations of tTF-NGR as measured by FX activation. This effect can be reverted by masking of PS with Annexin V. HT1080 human sarcoma xenografted tumors showed shrinkage induced by combined regional radiotherapy and systemic tTF-NGR as compared to growth inhibition achieved by either of the treatment modalities alone. CONCLUSIONS: Irradiation renders tumor and tumor vascular cells procoagulant by PS upregulation on their outer surface and radiotherapy can significantly improve the therapeutic antitumor efficacy of tTF-NGR in the xenograft model used. This synergistic effect will influence design of future clinical combination studies.
Asunto(s)
Antineoplásicos/farmacología , Antígenos CD13/metabolismo , Terapia Molecular Dirigida , Sarcoma/tratamiento farmacológico , Sarcoma/radioterapia , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/efectos de la radiación , Línea Celular Tumoral , Terapia Combinada , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Fosfatidilserinas/metabolismo , Sarcoma/metabolismo , Sarcoma/patologíaRESUMEN
BACKGROUND: Aminopeptidase N (CD13) is present on tumor vasculature cells and some tumor cells. Truncated tissue factor (tTF) with a C-terminal NGR-peptide (tTF-NGR) binds to CD13 and causes tumor vascular thrombosis with infarction. METHODS: We treated 17 patients with advanced cancer beyond standard therapies in a phase I study with tTF-NGR (1-h infusion, central venous access, 5 consecutive days, and rest periods of 2 weeks). The study allowed intraindividual dose escalations between cycles and established Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicity (DLT) by verification cohorts. RESULTS: MTD was 3 mg/m2 tTF-NGR/day × 5, q day 22. DLT was an isolated and reversible elevation of high sensitivity (hs) Troponin T hs without clinical sequelae. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were reversible upon anticoagulation. Imaging by contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) showed major tumor-specific reduction of blood flow in all measurable lesions as proof of principle for the mode of action of tTF-NGR. There were no responses as defined by Response Evaluation Criteria in Solid Tumors (RECIST), although some lesions showed intratumoral hemorrhage and necrosis after tTF-NGR application. Pharmacokinetic analysis showed a t1/2(terminal) of 8 to 9 h without accumulation in daily administrations. CONCLUSION: tTF-NGR is safely applicable with this regimen. Imaging showed selective reduction of tumor blood flow and intratumoral hemorrhage and necrosis.
RESUMEN
Truncated tissue factor (tTF)-NGR consists of the extracellular domain of the human TF and the binding motif NGR. tTF-NGR activates blood coagulation within the tumour vasculature following binding to CD13, and is overexpressed in the endothelial cells of tumour vessels, resulting in tumour vessel infarction and subsequent retardation/regression of tumour growth. The aim of the present study was to investigate gadofosveset-based real-time dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in evaluating the initial therapeutic effects of the anti-vascular tTF-NGR approach. DCE-MRI (3.0 T) was performed in human U87-glioblastoma tumour-bearing nude mice. During a dynamic T1w GE-sequence, a gadolinium-based blood pool contrast agent (gadofosveset) was injected via a tail vein catheter. Following the maximum contrast intensity inside the tumour being obtained, tTF-NGR was injected (controls received NaCl) and the contrast behaviour of the tumour was monitored by ROI analysis. The slope difference of signal intensities between controls and the tTF-NGR group was investigated, as well as the differences between the average area under the curve (AUC) of the two groups. The association between intensity, group (control vs. tTF-NGR group) and time was analysed by fitting a linear mixed model. Following the injection of tTF-NGR, the signal intensity inside the tumours exhibited a statistically significantly stronger average slope decrease compared with the signal intensity of the tumours in the NaCl group. Furthermore, the initial average AUC values of mice treated with tTF-NGR were 5.7% lower than the average AUC of the control animals (P<0.05). Gadofosveset-enhanced MRI enables the visualization of the initial tumour response to anti-vascular treatment in real-time. Considering the clinical application of tTF-NGR, this method may provide a simple alternative parameter for monitoring the tumour response to vascular disrupting agents and certain vascular targeting agents in humans.
RESUMEN
Calcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRLhigh AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR-Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML.