RESUMO
Pulmonary vascular remodeling is a key pathological process of pulmonary arterial hypertension (PAH), characterized by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Bortezomib (BTZ) is the first Food and Drug Administration (FDA)-approved proteasome inhibitor for multiple myeloma treatment. Recently, there is emerging evidence showing its effect on reversing PAH, although its mechanisms are not well understood. In this study, anti-proliferative and anti-migratory effects of BTZ on PASMCs were first examined by different inducers such as fetal bovine serum (FBS), angiotensin II (Ang II) and platelet-derived growth factor (PDGF)-BB, while potential mechanisms including cellular reactive oxygen species (ROS) and mitochondrial ROS were then investigated; finally, signal transduction of ERK and Akt was examined. Our results showed that BTZ attenuated FBS-, Ang II- and PDGF-BB-induced proliferation and migration, with associated decreased cellular ROS production and mitochondrial ROS production. In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB was also inhibited by BTZ treatment. This study indicates that BTZ can prevent proliferation and migration of PASMCs, which are possibly mediated by decreased ROS production and down-regulation of ERK and Akt. Thus, proteasome inhibition can be a novel pharmacological target in the management of PAH.
Assuntos
Bortezomib , Movimento Celular , Proliferação de Células , Miócitos de Músculo Liso , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-akt , Artéria Pulmonar , Espécies Reativas de Oxigênio , Bortezomib/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Angiotensina II/farmacologia , Becaplermina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Fosforilação/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismoRESUMO
Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/química , Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Estudo de Prova de Conceito , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/metabolismo , Células RAW 264.7 , RatosRESUMO
We have recently shown that exogenous administration of extracellular heat shock protein HSC70, a previously recognized intracellular chaperone protein, can protect against LPS-induced cardiac dysfunction through anti-inflammatory actions. However, whether it can also exert anti-hypertrophic effect is unknown. The present study was aimed to investigate the efficacy of HSC70 against cardiac hypertrophy and its underlying molecular mechanisms. Cardiomyocytes were isolated from the cardiac ventricles of neonatal Wistar rats and LPS (1 µg/mL) was used to induce the hypertrophic responses. We found that HSC70 (0.1, 1 and 5 µg/mL) pretreatment attenuated LPS-induced cardiomyocyte hypertrophy dose-dependently. In addition, HSC70 mitigated LPS-induced inflammatory mediators including TNF-α, IL-6, NO, iNOS and COX-2, with down-regulated protein expression of MMP-2 and MMP-9. Moreover, HSC70 repressed LPS-induced signaling of MAPK and Akt. Finally, HSC70 inhibited NF-κB subunit p65, and the DNA binding activity of NF-κB. Taken together, these findings suggest that in vitro HSC70 can exert anti-hypertrophic effects through inhibition of pro-inflammatory mediators, which are potential mediated by the down-regulation of MAPK, Akt and NF-κB signaling pathways. In conclusion, extracellular HSC70 may be a novel pharmacologic strategy in the management of cardiac hypertrophy.
Assuntos
Anti-Inflamatórios/farmacologia , Cardiomegalia/prevenção & controle , Proteínas de Choque Térmico HSC70/farmacologia , Lipopolissacarídeos/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Proteínas Recombinantes/farmacologia , Fator de Transcrição RelA/metabolismoRESUMO
Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine-citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody-drug conjugates.
Assuntos
Cisteína/química , Dissulfetos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Animais , Compostos de Boro , Monitoramento de Medicamentos/métodos , Endocitose , Endossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência/tendências , Humanos , Imunoconjugados , Lisossomos/metabolismo , Receptor ErbB-2/imunologia , RodaminasRESUMO
Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.
Assuntos
Transporte Biológico , Transferência Ressonante de Energia de Fluorescência , Imunoconjugados/farmacocinética , Permeabilidade da Membrana Celular , Reagentes de Ligações Cruzadas/química , Humanos , Imunoconjugados/metabolismo , PeptídeosRESUMO
Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.
Assuntos
Sistema Nervoso Central/fisiologia , Eletrofisiologia/métodos , Neuregulina-1/metabolismo , Esquizofrenia/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Modelos Animais de Doenças , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuregulina-1/genética , Neuregulina-1/imunologia , Estabilidade Proteica , Receptor ErbB-4/genética , Receptor ErbB-4/imunologia , Receptor ErbB-4/metabolismo , Risco , Esquizofrenia/genética , Transdução de Sinais , Transmissão SinápticaRESUMO
THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.
Assuntos
Antineoplásicos Imunológicos/química , Cisteína/química , Dissulfetos/química , Imunoconjugados/química , Maleimidas/química , Trastuzumab/química , Animais , Antineoplásicos Imunológicos/sangue , Cisteína/sangue , Cisteína/genética , Dissulfetos/sangue , Estabilidade de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Imunoconjugados/sangue , Maleimidas/sangue , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oligopeptídeos/sangue , Oligopeptídeos/química , Agregados Proteicos , Estabilidade Proteica , Ratos , Trastuzumab/sangue , Trastuzumab/genéticaRESUMO
Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
Assuntos
Catepsina B/metabolismo , Descoberta de Drogas , Imunoconjugados/química , Imunoconjugados/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Humanos , Espaço Intracelular/metabolismo , Especificidade por SubstratoRESUMO
PURPOSE: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. EXPERIMENTAL DESIGN: The F(ab')2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab')2) was generated and assessed in efficacy and toxicity studies. RESULTS: Anti-DLL4 F(ab')2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab')2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. CONCLUSIONS: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab')2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/farmacocinética , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macaca fascicularis , Camundongos , Ratos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Antibody-drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium-phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. EXPERIMENTAL DESIGN: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non-small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. RESULTS: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. CONCLUSIONS: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Imunoconjugados/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Oligopeptídeos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ratos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of (125)Iodide and (111)Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.
Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/imunologia , Área Sob a Curva , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Radioisótopos de Índio/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Radioisótopos do Iodo/farmacocinética , Neoplasias Pulmonares/imunologia , Proteínas de Membrana/imunologia , Taxa de Depuração Metabólica , Camundongos Nus , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines. This results in a heterogeneous mixture of antibody species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In order to characterize the assays used to quantitate total MC-vc-PAB-MMAE ADCs (conjugated and unconjugated antibody), we used purified fractions with defined DARs from 6 therapeutic antibodies to evaluate different assay formats and reagents. Our investigations revealed that for quantitation of total antibody, including all unconjugated and conjugated antibody species, sandwich ELISA formats did not always allow for recovery of all purified DAR fractions (DAR 0-8) to within ±20% of the expected values at the reagent concentrations tested. In evaluating alternative approaches, we found that the recovery of DAR fractions with semihomogeneous assay (SHA) formats, in which sample, capture, and detection reagents are preincubated in solution, were less affected by the antibody's MMAE drug load as compared to traditional stepwise sandwich ELISAs. Thus, choosing the optimal assay format and reagents for total antibody assays is valuable for developing accurate quantitative assays.
Assuntos
Antineoplásicos/farmacocinética , Imunotoxinas/farmacocinética , Oligopeptídeos/farmacocinética , Moduladores de Tubulina/farmacocinética , Animais , Antineoplásicos/química , Ensaio de Imunoadsorção Enzimática , Imunotoxinas/química , Camundongos , Camundongos SCID , Oligopeptídeos/química , Moduladores de Tubulina/químicaRESUMO
Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neuregulina-1/antagonistas & inibidores , Transdução de Sinais , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Humanos , Ligantes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/metabolismo , Neoplasia Residual/patologia , Neuregulina-1/metabolismo , Receptor ErbB-4 , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibodies against epidermal growth factor receptor (EGFR)--cetuximab and panitumumab--are widely used to treat colorectal cancer. Unfortunately, patients eventually develop resistance to these agents. We describe an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab. Cells with this mutation, however, retain binding to and are growth inhibited by panitumumab. Two of ten subjects studied here with disease progression after cetuximab treatment acquired this mutation. A subject with cetuximab resistance harboring the S492R mutation responded to treatment with panitumumab.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/genética , Epitopos/genética , Gefitinibe , Humanos , Mutação de Sentido Incorreto/genética , Panitumumabe , Quinazolinas/uso terapêuticoRESUMO
3A5 is a novel antibody that binds repeated epitopes within CA125, an ovarian tumor antigen that is shed into the circulation. Binding to shed antigen may limit the effectiveness of therapeutic antibodies because of unproductive immune complex (IC) formation and/or altered antibody distribution. To evaluate this possibility, we characterized the impact of shed CA125 on the in vivo distribution of 3A5. In vitro, 3A5 and CA125 were found to form ICs in a concentration-dependent manner. This phenomenon was then evaluated in vivo using quantitative whole-body autoradiography to assess the tissue distribution of (125)I-3A5 in an orthotopic OVCAR-3 tumor mouse model at different stages of tumor burden. Low doses of 3A5 (75 µg/kg) and pathophysiological levels of shed CA125 led to the formation of ICs in vivo that were rapidly distributed to the liver. Under these conditions, increased clearance of 3A5 from normal tissues was observed in mice bearing CA125-expressing tumors. Of importance, despite IC formation, 3A5 uptake by tumors was sustained over time. At a therapeutically relevant dose of 3A5 (3.5 mg/kg), IC formation was undetectable and distribution to normal tissues followed that of blood. In contrast, increased levels of radioactivity were observed in the tumors. These data demonstrate that CA125 and 3A5 do form ICs in vivo and that the liver is involved in their uptake. However, at therapeutic doses of 3A5 and clinically relevant CA125 levels, IC formation consumes only a minor fraction of 3A5, and tumor targeting seems to be unaffected.
Assuntos
Anticorpos/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Antígeno Ca-125/imunologia , Neoplasias Ovarianas/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Distribuição TecidualRESUMO
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.
Assuntos
Anticorpos Monoclonais/farmacologia , Glucose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteassoma , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Imunotoxinas/farmacocinética , Neoplasias Ovarianas/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/genética , Especificidade de Anticorpos , Sítios de Ligação , Antígeno Ca-125/imunologia , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Cisteína/genética , Feminino , Humanos , Macaca fascicularis , Proteínas de Membrana/imunologia , Camundongos , Mutagênese Sítio-Dirigida , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/farmacologiaRESUMO
BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.
Assuntos
Fator Ativador de Células B/imunologia , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/química , Sítios de Ligação , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic alpha-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2'-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for beta-catenin. Sequencing of the tumor DNA encoding beta-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.