RESUMO
Therapeutics at or close to the nanoscale, such as liposomal irinotecan, offer significant promise for the treatment of solid tumors. Their potential advantage over the unencapsulated or free form of the drug is due in part to their altered biodistribution. For slow and sustained release, significant optimization of formulation is needed to achieve the required level of stability and allow long-term storage of the drug product. Gradient-based liposomal formulation of camptothecins such as irinotecan poses unique challenges owing to the camptothecin- and acid-catalyzed hydrolysis of phospholipid esters in the inner monolayer of the liposomal membrane. We demonstrated that a narrow set of conditions related to the external pH, temperature, intraliposomal concentration, identity of the drug-trapping agent, physical form of the drug inside the liposomes, and final drug load have a marked impact on the stability of the liposome phospholipid membrane. The physical form of the drug inside the liposome was shown to be an insoluble gel with an irinotecan-to-sulfate ratio approximating 1:1, reducing the potential for irinotecan-catalyzed phospholipid hydrolysis in the internal phospholipid monolayer. As a result of this work, a stable and active liposome formulation has been developed that maintains phospholipid chemical stability following long-term storage at 2-8°C.
Assuntos
Lipossomos , Fosfolipídeos , Irinotecano , Estabilidade de Medicamentos , Distribuição Tecidual , Camptotecina , CatáliseRESUMO
Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.
Assuntos
Nanopartículas , Anticorpos de Cadeia Única , Dissulfetos/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Nanopartículas/químicaRESUMO
Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.
RESUMO
Effective liposomal formulations of vinorelbine (5' nor-anhydro-vinblastine; VRL) have been elusive due to vinorelbine's hydrophobic structure and resulting difficulty in stabilizing the drug inside the nanocarrier. Triethylammonium salts of several polyanionic trapping agents were used initially to prepare minimally pegylated nanoliposomal vinorelbine formulations with a wide range of drug release rates. Sulfate, poly(phosphate), and sucrose octasulfate were used to stabilize vinorelbine intraliposomally while in circulation, with varying degrees of effectiveness. The release rate of vinorelbine from the liposomal carrier was affected by both the chemical nature of the trapping agent and the resulting drug-to-lipid ratio, with liposomes prepared using sucrose octasulfate displaying the longest half-life in circulation (9.4 h) and in vivo retention in the nanoparticle (t(1/2) = 27.2 h). Efficacy was considerably improved in both a human colon carcinoma (HT-29) and a murine (C-26) colon carcinoma model when vinorelbine was stably encapsulated in liposomes using triethylammonium sucrose octasulfate. Early difficulties in preparing highly pegylated formulations were later overcome by substituting a neutral distearoylglycerol anchor for the more commonly used anionic distearoylphosphatidylethanolamine anchor. The new pegylated nanoliposomal vinorelbine displayed high encapsulation efficiency and in vivo drug retention, and it was highly active against human breast and lung tumor xenografts. Acute toxicity of the drug in immunocompetent mice slightly decreased upon encapsulation in liposomes, with a maximum tolerated dose of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Our results demonstrate that a highly active, stable, and long-circulating liposomal vinorelbine can be prepared and warrants further study in the treatment of cancer.
Assuntos
Vimblastina/análogos & derivados , Portadores de Fármacos , Estabilidade de Medicamentos , Humanos , Lipossomos , Nanopartículas , Fosfolipídeos/sangue , Trítio , Vimblastina/química , Vimblastina/farmacocinética , VinorelbinaRESUMO
Mesothelioma is a malignancy of the mesothelium and current treatments are generally ineffective. One promising area of anticancer drug development is to explore tumor susceptibility to targeted therapy. To achieve efficient, targeted intracellular delivery of therapeutic agents to mesothelioma cells, we selected a naive human single-chain (scFv) phage antibody display library directly on the surface of live mesothelioma cells to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. We have identified a panel of internalizing scFvs that bind to mesothelioma cell lines derived from both epithelioid (M28) and sarcomatous (VAMT-1) types of this disease. Most importantly, these antibodies stain mesothelioma cells in situ and therefore define a panel of clinically represented tumor antigens. We have further exploited the internalizing function of these scFvs to achieve targeted intracellular drug delivery to mesothelioma cells. We showed that scFv-targeted immunoliposomes were efficiently and specifically taken up by both epithelioid and sarcomatous mesothelioma cells, but not control cells, and immunoliposomes encapsulating the small-molecule drug topotecan caused targeted killing of both types of mesothelioma cells in vitro.
Assuntos
Anticorpos/imunologia , Antineoplásicos/uso terapêutico , Fragmentos de Imunoglobulinas/imunologia , Mesotelioma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Humanos , Mesotelioma/imunologiaRESUMO
Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114â¯mol% taxane per mole phospholipid) and subsequent stability (>3â¯years at 2-8⯰C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69â¯nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12â¯h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50â¯mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10â¯mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).
Assuntos
Antineoplásicos/administração & dosagem , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Portadores de Fármacos/química , Efrina-A2/metabolismo , Nanopartículas/química , Pró-Fármacos/administração & dosagem , Taxoides/administração & dosagem , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Humanos , Lipossomos , Camundongos Nus , Tamanho da Partícula , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ligação Proteica , Taxoides/química , Taxoides/farmacocinética , Taxoides/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Nanopartículas/uso terapêutico , Receptor EphA2/metabolismo , Animais , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Docetaxel/sangue , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/uso terapêutico , Humanos , Lipossomos , Camundongos Endogâmicos NOD , Camundongos SCID , Taxoides/farmacologia , Taxoides/uso terapêutico , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.
Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Nanopartículas , Neoplasias/imunologia , Neoplasias/metabolismo , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Regulação da Expressão Gênica , Humanos , Lipossomos/imunologia , Dados de Sequência Molecular , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Alinhamento de Sequência , Solubilidade , Topotecan/toxicidadeRESUMO
OBJECT: Many factors relating to the safety and efficacy of convection-enhanced delivery (CED) into intracranial tumors are poorly understood. To investigate these factors further and establish a more clinically relevant large animal model, with the potential to investigate CED in large, spontaneous tumors, the authors developed a magnetic resonance (MR) imaging-compatible system for CED of liposomal nanoparticles into the canine brain, incorporating real-time MR imaging. Additionally any possible toxicity of liposomes containing Gd and the chemotherapeutic agent irinotecan (CPT-11) was assessed following direct intraparenchymal delivery. METHODS: Four healthy laboratory dogs were infused with liposomes containing Gd, rhodamine, or CPT-11. Convection-enhanced delivery was monitored in real time by sequential MR imaging, and the volumes of distribution were calculated from MR images and histological sections. Assessment of any toxicity was based on clinical and histopathological evaluation. Convection-enhanced delivery resulted in robust volumes of distribution in both gray and white matter, and real-time MR imaging allowed accurate calculation of volumes and pathways of distribution. RESULTS: Infusion variability was greatest in the gray matter, and was associated with leakage into ventricular or subarachnoid spaces. Complications were minimal and included mild transient proprioceptive deficits, focal hemorrhage in 1 dog, and focal, mild perivascular, nonsuppurative encephalitis in 1 dog. CONCLUSIONS: Convection-enhanced delivery of liposomal Gd/CPT-11 is associated with minimal adverse effects in a large animal model, and further assessment for use in clinical patients is warranted. Future studies investigating real-time monitored CED in spontaneous gliomas in canines are feasible and will provide a unique, clinically relevant large animal translational model for testing this and other therapeutic strategies.
Assuntos
Camptotecina/análogos & derivados , Imageamento por Ressonância Magnética , Animais , Encéfalo/metabolismo , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/toxicidade , Cães , Monitoramento Ambiental , Feminino , Fluorescência , Gadolínio , Irinotecano , Lipossomos , NanopartículasRESUMO
To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/isolamento & purificação , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/genética , Especificidade de Anticorpos/imunologia , Antígenos de Neoplasias/química , Células CHO , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida , Cricetinae , Cricetulus , Regulação para Baixo , Endocitose , Epitopos/imunologia , Humanos , Região Variável de Imunoglobulina/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Fenótipo , Ligação ProteicaRESUMO
Liposome formulations of camptothecins have been actively pursued because of the potential for significant pharmacologic advantages from successful drug delivery of this important class of anticancer drugs. We describe nanoliposomal CPT-11, a novel nanoparticle/liposome construct containing CPT-11 (irinotecan) with unprecedented drug loading efficiency and in vivo drug retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or nonpolymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (>800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 hours. CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. Nanoliposomal CPT-11 showed markedly superior efficacy when compared with free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models. This study shows that intraliposomal stabilization of CPT-11 using a polymeric or highly charged, nonpolymeric polyanionic trapping agent results in a markedly active antitumor agent with low toxicity.
Assuntos
Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Nanoestruturas/química , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Camptotecina/administração & dosagem , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/toxicidade , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Estabilidade de Medicamentos , Feminino , Células HT29 , Humanos , Irinotecano , Lipossomos/administração & dosagem , Lipossomos/farmacocinética , Lipossomos/toxicidade , Camundongos , Camundongos Nus , Nanoestruturas/toxicidade , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 microg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t(1/2)) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t(1/2) = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t(1/2) by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t(1/2) = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 x 10(-5)) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Animais , Neoplasias Encefálicas/metabolismo , Camptotecina/administração & dosagem , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/toxicidade , Linhagem Celular Tumoral , Convecção , Humanos , Irinotecano , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/farmacocinética , Lipossomos/toxicidade , Masculino , Nanoestruturas/química , Nanoestruturas/toxicidade , Fosfolipídeos/administração & dosagem , Fosfolipídeos/química , Fosfolipídeos/farmacocinética , Fosfolipídeos/toxicidade , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.
Assuntos
Neoplasias da Mama/imunologia , Lipossomos/imunologia , Lipossomos/farmacocinética , Nanoestruturas , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Humanos , Lipossomos/química , Camundongos , Receptor ErbB-2/biossíntese , Transplante HeterólogoRESUMO
Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.
Assuntos
Molécula de Adesão de Leucócito Ativado/imunologia , Anticorpos Monoclonais/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias da Próstata/tratamento farmacológico , Antibióticos Antineoplásicos/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Comunicação Celular/imunologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Citometria de Fluxo , Humanos , Fragmentos de Imunoglobulinas/imunologia , Ligantes , Lipossomos , Masculino , Neoplasias da Próstata/patologia , Radiossensibilizantes/administração & dosagem , Topotecan/administração & dosagem , Vimblastina/administração & dosagem , Vimblastina/análogos & derivados , VinorelbinaRESUMO
We have previously shown that convection-enhanced delivery (CED) of highly stable nanoparticle/liposome agents encapsulating chemotherapeutic drugs is effective against intracranial rodent brain tumor xenografts. In this study, we have evaluated the combination of a newly developed nanoparticle/liposome containing the topoisomerase I inhibitor CPT-11 (nanoliposomal CPT-11 [nLs-CPT-11]), and PEGylated liposomal doxorubicin (Doxil) containing the topoisomerase II inhibitor doxorubicin. Both drugs were detectable in the CNS for more than 36 days after a single CED application. Tissue half-life was 16.7 days for nLs-CPT-11 and 10.9 days for Doxil. The combination of the two agents produced synergistic cytotoxicity in vitro. In vivo in U251MG and U87MG intracranial rodent xenograft models, CED of the combination was also more efficacious than either agent used singly. Analysis of the parameters involved in this approach indicated that tissue pharmacokinetics, tumor microanatomy, and biochemical interactions of the drugs all contributed to the therapeutic efficacy observed. These findings have implications for further clinical applications of CED-based treatment of brain tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Doxorrubicina/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/farmacocinética , Linhagem Celular Tumoral , Convecção , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Sinergismo Farmacológico , Meia-Vida , Humanos , Irinotecano , Lipossomos , Masculino , Nanopartículas , Transplante de Neoplasias , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Imunoconjugados/uso terapêutico , Lipossomos/administração & dosagem , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Cetuximab , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Epirubicina/administração & dosagem , Receptores ErbB/genética , Receptores ErbB/imunologia , Feminino , Glioblastoma/imunologia , Glioblastoma/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Vimblastina/administração & dosagem , Vimblastina/análogos & derivados , VinorelbinaRESUMO
Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Efrina-A2/imunologia , Imunoconjugados/imunologia , Nanopartículas , Anticorpos de Cadeia Única/imunologia , Animais , Humanos , Região Variável de Imunoglobulina/imunologia , Engenharia de Proteínas/métodos , Estabilidade Proteica , Receptor EphA2 , Anticorpos de Cadeia Única/biossínteseRESUMO
Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies.Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797).Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52-18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42).Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190-202. ©2017 AACR.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Nanopartículas/administração & dosagem , Adolescente , Adulto , Idoso , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos da radiação , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/química , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Feminino , Humanos , Fígado/diagnóstico por imagem , Fígado/efeitos dos fármacos , Pessoa de Meia-Idade , Nanopartículas/química , Metástase Neoplásica , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptor ErbB-2/sangue , Baço/diagnóstico por imagem , Baço/patologia , Trastuzumab/administração & dosagemRESUMO
BACKGROUND: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. RESULTS: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. CONCLUSION: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
Assuntos
Anticorpos Antineoplásicos/administração & dosagem , Anticorpos Antineoplásicos/análise , Técnicas Imunológicas , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Antineoplásicos/metabolismo , Morte Celular , Linhagem Celular Tumoral , Doxorrubicina/química , Histidina/metabolismo , Humanos , Região Variável de Imunoglobulina/imunologia , Ligantes , Bicamadas Lipídicas/química , Metabolismo dos Lipídeos , Metotrexato/química , Níquel/metabolismo , Oligopeptídeos/metabolismo , Receptor ErbB-2/imunologia , Sensibilidade e EspecificidadeRESUMO
Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t1/2 = 1.5 days), whereas free topotecan was rapidly cleared (t1/2 = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.