RESUMO
Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine.
RESUMO
We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.
Assuntos
Neoplasias da Mama , Nanopartículas Metálicas , Trastuzumab , Feminino , Humanos , Neoplasias da Mama/diagnóstico , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Trastuzumab/químicaRESUMO
Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.
Assuntos
Anticorpos Monoclonais , Dextranos , Animais , Cricetinae , Hidrogéis/química , Cricetulus , Peptídeos/química , Trastuzumab/químicaRESUMO
Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.
Assuntos
Fucose , Fucosiltransferases , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Fucose/metabolismo , Fucosiltransferases/genética , Imunoglobulina G/química , PolissacarídeosRESUMO
The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.
Assuntos
Antineoplásicos Imunológicos , Anidrases Carbônicas , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Biomarcadores Tumorais , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Linhagem Celular Tumoral , Humanos , Concentração de Íons de HidrogênioRESUMO
The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.
Assuntos
Adjuvantes Imunológicos/química , Antígenos Arqueais/química , Vacinas contra COVID-19/uso terapêutico , Lipídeos/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Peso Corporal , COVID-19/terapia , Chlorocebus aethiops , Cricetinae , Citocinas/metabolismo , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Passiva , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Peptídeos/química , Domínios Proteicos , SARS-CoV-2 , Receptores Toll-Like/imunologia , Células Vero , Carga Viral , Soroterapia para COVID-19RESUMO
Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.
Assuntos
Afinidade de Anticorpos/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Células CHO , Camelídeos Americanos , Morte Celular , Linhagem Celular Tumoral , Cricetulus , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cinética , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores Proteína Tirosina Quinases/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.
Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/sangue , Área Sob a Curva , COVID-19 , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva/métodos , Testes de Neutralização , Pandemias , Análise de Regressão , Estudos de Amostragem , Resultado do Tratamento , Proteínas do Envelope Viral/imunologia , Soroterapia para COVID-19RESUMO
Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic Abs for cancer.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/terapia , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/genética , Adenocarcinoma/imunologia , Animais , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/imunologia , Camelidae , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação/genética , Ligação Proteica , Engenharia de ProteínasRESUMO
The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG1 (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin. We showed that the IGF-TRAP had potent anti-cancer activity in several pre-clinical models of aggressive carcinomas. Here we report on the re-engineering of the IGF-TRAP with the aim of improving physicochemical properties and suitability for clinical applications. We show that cysteine-serine substitutions in the Fc hinge region of IGF-TRAP eliminated high-molecular-weight oligomerized species, while a further addition of a flexible linker, not only improved the pharmacokinetic profile, but also enhanced the therapeutic profile of the IGF-TRAP, as evaluated in an experimental colon carcinoma metastasis model. Dose-response profiles of the modified IGF-TRAPs correlated with their bio-availability profiles, as measured by the IGF kinase-receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. This study provides a compelling example of structure-based re-engineering of Fc-fusion-based biologics for better manufacturability that also significantly improved pharmacological parameters. It identifies the re-engineered IGF-TRAP as a potent anti-cancer therapeutic.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Produtos Biológicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Feminino , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismoRESUMO
Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20-30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.
Assuntos
Anticorpos/uso terapêutico , Desenho de Fármacos , Proteínas Recombinantes/uso terapêutico , Afinidade de Anticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Mutação/genética , Ressonância de Plasmônio de Superfície , Termodinâmica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In an effort to rationalize and optimize an antiapoptotic coating combining chondroitin sulfate (CS) and epidermal growth factor (EGF) for vascular applications, the authors here report the comparison of two grafting strategies aiming to display EGF in an oriented fashion on CS. For that purpose, the authors produced, purified, and characterized a chimeric protein corresponding to EGF that was N-terminally fused to a cysteine and a coil peptide. The chimera was covalently immobilized via its free thiol group or captured via coiled-coil interactions at the surface of a biosensor or on a chondroitin sulfate coating in multiwell plates, mimicking the coating that was previously developed by them for stent-graft surfaces. The interactions of grafted EGF with the soluble domain of its receptor or the impact of grafted EGF upon vascular smooth muscle survival in proapoptotic conditions indicated that the coiled-coil based tethering was the best approach to display EGF. These results, combined to direct enzyme-linked immunosorbent assay measurements, indicated that the coiled-coil tethering approach allowed increasing the amount of bioavailable EGF when compared to covalent coupling, rather than the total amount of grafted EGF, while using much lower concentrations of tagged EGF during incubation.
Assuntos
Sulfatos de Condroitina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacocinética , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacocinética , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ligação Proteica , RatosRESUMO
Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a â¼75% improvement to peak cell densities.
Assuntos
Metabolômica/métodos , Proteômica/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Meios de Cultura , Humanos , Focalização Isoelétrica , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial BidimensionalRESUMO
Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression.
Assuntos
Expressão Gênica , Polietilenoimina/metabolismo , Acilação , Animais , Anticorpos/metabolismo , Células CHO , Morte Celular , Cricetinae , Cricetulus , DNA/metabolismo , Endocitose , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Mamíferos , Espectroscopia de Prótons por Ressonância Magnética , TransfecçãoRESUMO
Polymeric nanocarriers are attractive nonviral vectors for gene delivery purposes in vivo. For such applications, numerous physiological and subcellular bottlenecks have to be overcome. In that endeavor, each structural feature of nanocarriers can be optimized with respect to its corresponding challenges. Here, we focused on the interface between a model gene delivery nanocarrier and relevant constituents of the physiological environment. We screened a library of carboxymethylated dextrans (CMD) for the electrostatic coating of positively charged nanocarriers. We evaluated the jointed influence of the CMD molecular weight and charge density upon nanocarrier coating with respect to DNase, small ions, plasma proteins, red blood cells, and target cells. A total of 4 out of 26 CMD coated nanocarriers successfully passed every screening assay, but did not yield increased reporter gene expression in target cells compared to uncoated nanocarriers. The fine-tuning of CMD for nanocarrier coating yielded a relevant shortlist of candidates that will be further tested in vivo.
Assuntos
Dextranos/química , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Nanocápsulas/química , Animais , Linhagem Celular Tumoral , Dextranos/efeitos adversos , Eritrócitos/efeitos dos fármacos , Humanos , Ovinos , Eletricidade EstáticaRESUMO
The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/metabolismo , Carcinoma/patologia , Receptor IGF Tipo 1/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Especificidade de Anticorpos , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Células CHO , Carcinoma/tratamento farmacológico , Carcinoma/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Modelos Animais de Doenças , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Cinética , Camundongos , Metástase Neoplásica , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn. RESULTS: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin. CONCLUSIONS: These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.
Assuntos
Isótopos de Carbono/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Análise do Fluxo Metabólico/métodos , Fibras Musculares Esqueléticas/metabolismo , Resistina/metabolismo , Software , Animais , Biologia Computacional , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Desidrogenase (Lipoamida)/metabolismo , RatosRESUMO
Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.
Assuntos
Apoptose , Caspase 1/metabolismo , Proteína HMGB1/química , Receptores Imunológicos/metabolismo , Animais , Candida/metabolismo , Candidíase/imunologia , Células Dendríticas/microbiologia , Fibroblastos/citologia , Tolerância Imunológica , Imunidade Inata , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/química , Receptor para Produtos Finais de Glicação Avançada , Proteínas Recombinantes/metabolismo , Sepse/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis.
Assuntos
Citrobacter rodentium/fisiologia , Diarreia/metabolismo , Diarreia/microbiologia , Suscetibilidade a Doenças/microbiologia , Infecções por Enterobacteriaceae/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Animais , Diferenciação Celular , Mapeamento Cromossômico , Clonagem Molecular , Colo/microbiologia , Colo/patologia , Diarreia/patologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Estudos de Associação Genética , Loci Gênicos/genética , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos , Microvilosidades/microbiologia , Microvilosidades/patologia , Modelos Biológicos , Células Estromais/metabolismo , Células Estromais/microbiologia , Células Estromais/patologia , Análise de Sobrevida , Via de Sinalização WntRESUMO
Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.