RESUMO
MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Imunoconjugados/química , Lipossomos/química , Anticorpos de Cadeia Única/química , Animais , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Camundongos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Receptor ErbB-2/metabolismo , Microambiente Tumoral/efeitos dos fármacosRESUMO
Despite the advances in imaging, surgery and radiotherapy, the majority of patients with brainstem gliomas die within 2 years after initial diagnosis. Factors that contribute to the dismal prognosis of these patients include the infiltrative nature and anatomic location in an eloquent area of the brain, which prevents total surgical resection and the presence of the blood-brain barrier (BBB), which reduces the distribution of systemically administered agents. The development of new therapeutic approaches which can circumvent the BBB is a potential path to improve outcomes for these children. Convection-enhanced delivery (CED) and intranasal delivery (IND) are strategies that permit direct drug delivery into the central nervous system and are an alternative to intravenous injection (IV). We treated rats bearing human brainstem tumor xenografts with nanoliposomal irinotecan (CPT-11) using CED, IND, and IV. A single treatment of CED irinotecan had a similar effect on overall survival as multiple treatments by IV route. IND CPT-11 showed significantly increased survival of animals with brainstem tumors, and demonstrated the promise of this non-invasive approach of drug delivery bypassing the BBB when combined with nanoliposomal chemotherapy. Our results indicated that using CED and IND of nanoliposomal therapy increase likelihood of practical therapeutic approach for the treatment of brainstem gliomas.
Assuntos
Neoplasias do Tronco Encefálico/tratamento farmacológico , Irinotecano/administração & dosagem , Inibidores da Topoisomerase I/administração & dosagem , Administração Intranasal , Animais , Neoplasias do Tronco Encefálico/mortalidade , Linhagem Celular Tumoral , Convecção , Portadores de Fármacos , Humanos , Irinotecano/farmacocinética , Lipossomos , Masculino , Nanoestruturas , Ratos , Inibidores da Topoisomerase I/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liposomal irinotecan (irinotecan liposome injection, nal-IRI), a liposomal formulation of irinotecan, is designed for extended circulation relative to irinotecan and for exploiting discontinuous tumor vasculature for enhanced drug delivery to tumors. Following tumor deposition, nal-IRI is taken up by phagocytic cells followed by irinotecan release and conversion to its active metabolite, SN-38. Sustained inhibition of topoisomerase 1 by extended SN-38 exposure as a result of delivery by nal-IRI is hypothesized to enable superior antitumor activity compared with traditional topoisomerase 1 inhibitors such as conventional irinotecan and topotecan. We evaluated the antitumor activity of nal-IRI compared with irinotecan and topotecan in preclinical models of small-cell lung cancer (SCLC) including in a model pretreated with carboplatin and etoposide, a first-line regimen used in SCLC. Nal-IRI demonstrated antitumor activity in xenograft models of SCLC at clinically relevant dose levels, and resulted in complete or partial responses in DMS-53, DMS-114, and NCI-H1048 cell line-derived models as well as in three patient-derived xenograft models. The antitumor activity of nal-IRI was superior to that of topotecan in all models tested, which generally exhibited limited control of tumor growth and was superior to irinotecan in four out of five models. Further, nal-IRI demonstrated antitumor activity in tumors that progressed following treatment with topotecan or irinotecan, and demonstrated significantly greater antitumor activity than both topotecan and irinotecan in NCI-H1048 tumors that had progressed on previous carboplatin plus etoposide treatment. These results support the clinical development of nal-IRI in patients with SCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/análogos & derivados , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Inibidores da Topoisomerase I/administração & dosagem , Animais , Camptotecina/administração & dosagem , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Feminino , Humanos , Irinotecano , Lipossomos/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Distribuição Aleatória , Carcinoma de Pequenas Células do Pulmão/enzimologia , Topotecan/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/toxicidade , Neoplasias da Mama/patologia , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Nus , Miócitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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
Combinations of chemotherapy with immunotherapy have seen recent clinical success, including two approvals of anti-PD-1/L1 agents in combination with taxane-based chemotherapy in non-small cell lung cancer and triple-negative breast cancer. Here, we present a study on the combination activity and mechanistic rationale of a novel EphA2-targeted liposomal taxane (EphA2-ILs-DTXp) and anti-PD-1. This combination was highly active in mouse syngeneic tumor models, with complete responses observed in 3 of 5 models. In the EMT-6 tumor model, combination of EphA2-ILs-DTXp with anti-PD-1 resulted in a 60% complete response rate, with durable responses that were resistant to rechallenge. These responses were not observed in the absence of CD8+ T cells. Characterization of the immune infiltrates in EMT-6 tumors reveals increased CD8+ T cells, increased CD8+ IFNγ+ CTLs, and an increased CD8/regulatory T-cell (Treg) ratio. These immunomodulatory effects were not observed in mice treated with a combination of docetaxel and anti-PD-1. Pharmacokinetic analysis revealed that the AUC of docetaxel was increased 15 times, from 52.1 to 785 ng/mL/hour, when delivered by EphA2-ILs-DTXp. A dose reduction study of EphA2-ILs-DTXp showed a dose-response relationship for both tumor growth inhibition and the CD8/Treg ratio. Our data indicate that synergism between docetaxel and anti-PD-1 is achievable with nanoliposomal delivery.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor EphA2/metabolismo , Taxoides/uso terapêutico , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias/patologia , Taxoides/farmacologiaRESUMO
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
To guide developers of innovative and generic drug products that contain nanomaterials, the U.S. Food and Drug Administration issued the draft guidance for industry titled: "Drug Products, Including Biological Products, that Contain Nanomaterials" in December 2017. During the AAPS Guidance Forum on September 11, 2018, participants from industry, academia, and regulatory bodies discussed this draft guidance in an open setting. Two questions raised by the AAPS membership were discussed in more detail: what is the appropriate regulatory pathway for approval of drug products containing nanomaterials, and how to determine critical quality attributes (CQAs) for nanomaterials? During the meeting, clarification was provided on how the new FDA center-led guidance relates to older, specific nanomaterial class, or specific product-related guidances. The lively discussions concluded with some clear observations and recommendations: (I) Important lessons can be learned from how CQAs were determined for, e.g., biologics. (II) Publication of ongoing scientific discussions on strategies and studies determining CQAs of drug products containing nanomaterials will significantly strengthen the science base on this topic. Furthermore, (III) alignment on a global level on how to address new questions regarding nanomedicine development protocols will add to efficient development and approval of these much needed candidate nanomedicines (innovative and generic). Public meetings such as the AAPS Guidance Forum may serve as the place to have these discussions.
Assuntos
Produtos Biológicos/normas , Indústria Farmacêutica/normas , Medicamentos Genéricos/normas , Guias como Assunto , Nanoestruturas/normas , Aprovação de Drogas/legislação & jurisprudência , Indústria Farmacêutica/legislação & jurisprudência , Regulamentação Governamental , Estados Unidos , United States Food and Drug AdministrationRESUMO
Therapeutically targeting receptor tyrosine kinases has proven to be paramount to overcoming chemotherapy resistance in several cancer indications, improving patient outcomes. Insulin-Like Growth Factor Receptor 1 (IGF-1R) and Epidermal Growth Factor Receptor 3 (ErbB3) have been implicated as two such drivers of resistance, however their simultaneous role in ovarian cancer chemotherapy resistance remains poorly elucidated. The aim of this work is to determine the effects of dual IGF-1R/ErbB3 inhibition on ovarian cancer cell signaling, growth, and in vivo efficacy. Assessment of in vitro chemotherapy response across a panel of ovarian cancer cell lines revealed that increased IGF-1R cell surface expression correlates with decreased sensitivity to chemotherapy, and that growth induced by IGF-1R and ErbB3 ligands is blocked by the tetravalent bispecific antibody targeting IGF-1R and ErbB3, istiratumab. In vitro chemotherapy treatment increased ovarian cancer cell line capacity to activate prosurvival PI3K signaling in response to ligand, which could be prevented with istiratumab treatment. Furthermore, in vivo efficacy of standard of care chemotherapies using a xenograft model of ovarian cancer was potentiated with istiratumab. Our results suggest a role for IGF-1R and ErbB3 in driving chemotherapy resistance of ovarian cancer.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Receptor ErbB-3/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Paclitaxel/farmacologia , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Receptor ErbB-3/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , 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
Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Animais , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/terapia , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
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
We have used a naive human single-chain fragment variable (scFv) library as a source of random shape repertoire to directly probe the altered surface chemistry of tumor cells. We reported previously the identification of more than 90 internalizing phage monoclonal antibodies targeting prostate cancer cells, including those that are hormone refractory. In this report, we describe the conversion of a panel of those scFvs into full-length human immunoglobulins (IgGs) and show that tumor specificity is retained. We have further shown that antibodies isolated from a naive phage display library can nevertheless be of high affinity towards target tumor cells. In addition, full-length IgGs retain the functionality of parental scFvs including the ability to rapidly enter target cells through receptor-mediated endocytosis and thereby to mediate efficient and specific intracellular payload delivery to tumor cells. We have used recombinant IgGs to immunoprecipitate target antigens and analyzed their molecular composition by mass spectrometry. We have identified one target antigen as activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166 and have further studied tissue specificity of this internalizing ALCAM epitope by immunohistochemistry. Our study shows that cell type-specific internalizing human antibody can be readily identified from a naive phage antibody display library, characterized with regards to sequence, affinity, tissue specificity, and antigen identity, and modified genetically and chemically to generate various forms of targeted therapeutics.
Assuntos
Anticorpos Antineoplásicos/imunologia , Carcinoma/imunologia , Imunoglobulina G/imunologia , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/imunologia , Molécula de Adesão de Leucócito Ativado/imunologia , Molécula de Adesão de Leucócito Ativado/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/administração & dosagem , Células CHO , Carcinoma/patologia , Carcinoma/terapia , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Endocitose/efeitos dos fármacos , Humanos , Imunoglobulina G/administração & dosagem , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Imuno-Histoquímica , Imunoprecipitação , Imunoterapia/métodos , Lipossomos , Masculino , Espectrometria de Massas , Biblioteca de Peptídeos , Antígeno Prostático Específico/isolamento & purificação , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
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.