RESUMO
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
Assuntos
Biomarcadores , Antígeno CD47/química , Antígeno CD47/metabolismo , Proteínas de Transporte/metabolismo , Receptores Imunológicos/metabolismo , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/farmacologia , Antígenos de Diferenciação/imunologia , Sítios de Ligação , Antígeno CD47/efeitos dos fármacos , Antígeno CD47/genética , Humanos , Macrófagos/metabolismo , Modelos Moleculares , Fagocitose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Bloqueadores/química , COVID-19 , Córtex Cerebral , Neurônios , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , COVID-19/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Organoides/metabolismo , Organoides/patologia , Organoides/virologiaRESUMO
Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.
Assuntos
Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Proteína Acessória do Receptor de Interleucina-1/química , Interleucina-1beta/química , Modelos Moleculares , Conformação Proteica , Receptores Tipo I de Interleucina-1/química , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Humanos , Imunoglobulina G/química , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Betacoronavirus/imunologia , Enzima de Conversão de Angiotensina 2 , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , COVID-19 , Infecções por Coronavirus/imunologia , Cristalografia por Raios X , Epitopos de Linfócito B , Células HEK293 , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , SARS-CoV-2 , Hipermutação Somática de Imunoglobulina , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.
Assuntos
Tecido Adiposo/metabolismo , Anticorpos Bloqueadores/imunologia , Osso e Ossos/metabolismo , Epitopos , Hormônio Foliculoestimulante/imunologia , Animais , Anticorpos Bloqueadores/química , Anticorpos Monoclonais , Densidade Óssea , Feminino , Hormônio Foliculoestimulante/química , Subunidade beta do Hormônio Folículoestimulante/imunologia , Humanos , Hipercolesterolemia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Obesidade , Osteoporose , Receptores do FSH/metabolismoRESUMO
Norovirus is the causing agent of acute gastroenteritis disease globally. Efforts in developing therapeutics against virus infection mostly fail due to emergence of drug resistance that is a consequence of presence of high mutation rates in virus genome during virus' life cycle. In this study, we computationally analyzed the affinity of a drug target, wild type VP1 envelope protein and its three variants to a therapeutic antibody FAB5I2. We have found that mutations break important hydrogen bonds and cause high fluctuations in residues that form VP1-FAB5I2 complex interface. In addition to changes in dynamics, we also revealed that the affinity of FAB5I2 to VP1 protein drops significantly upon mutations in terms of relative binding free energy.
Assuntos
Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Antígenos Virais/química , Simulação de Dinâmica Molecular , Mutação , Norovirus/química , Proteínas do Envelope Viral/química , Anticorpos Bloqueadores/genética , Antígenos Virais/genética , Humanos , Norovirus/genética , Proteínas do Envelope Viral/genéticaRESUMO
Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR+ CXC chemokines. The engineered molecules recognize functional epitopes of ELR+ CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.
Assuntos
Anticorpos Bloqueadores/química , Artrite/terapia , Quimiocinas/metabolismo , Evolução Molecular Direcionada , Inflamação , Animais , Artrite/imunologia , Autoanticorpos/química , Ligação Competitiva , Biotinilação , Cálcio/química , Mapeamento de Epitopos , Epitopos/química , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Neutrófilos/citologia , Neutrófilos/metabolismo , Ligação Proteica , Transdução de Sinais , Propriedades de Superfície , Líquido Sinovial/metabolismo , TransgenesRESUMO
Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand binding and those due to structural rearrangements. Here, we used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Using maltose-binding protein (MBP) as a model system, customized phage-display selections were performed to generate sABs that stabilize MBP in different conformational states, modulating ligand-binding affinity in competitive, allosteric, or peristeric manners. We determined that the binding of a closed conformation-specific sAB (sAB-11M) to MBP in the absence of maltose is entropically driven, providing new insight into designing antibody-stabilized protein interactions. Crystal structures of sABs bound to MBP, together with biophysical data, delineate the basis of free energy differences between different conformational states and confirm the use of the sABs as energy probes for dissecting enthalpic and entropic contributions to conformational transitions. Our work provides a foundation for investigating the energetic contributions of distinct conformational dynamics to specific biological outputs. We anticipate that our approach also may be valuable for analyzing the energy landscapes of regulatory proteins controlling biological responses to environmental changes.
Assuntos
Anticorpos Bloqueadores/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Maltose/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Substituição de Aminoácidos , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/genética , Afinidade de Anticorpos , Apoproteínas/química , Apoproteínas/metabolismo , Biotinilação , Cristalografia por Raios X , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ligantes , Maltose/química , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Sondas Moleculares/química , Sondas Moleculares/genética , Mutação , Biblioteca de Peptídeos , Conformação Proteica , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
PURPOSE: Inhibitors of the ActRII signaling pathways represent promising therapeutics for the treatment of muscular diseases, but also pose risks as performance-enhancing agents in sports. Bimagrumab is a human anti-ActRII antibody which was found to increase muscle mass and function by blocking ActRII signaling. As it has considerable potential for being misused as doping agent in sports, the aim of this study was to develop a mass spectrometric detection assay for doping control serum samples. EXPERIMENTAL DESIGN: Within this study, a detection method for Bimagrumab in human serum was developed, which combines ammonium sulfate precipitation and affinity purification with proteolytic digestion and LC-HRMS. To facilitate the unambiguous identification of the diagnostic peptides, an orthogonal IM separation was additionally performed. RESULTS: The assay was successfully validated and the analysis of clinical samples demonstrated its fitness for purpose for an application in routine doping control analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Although no myostatin inhibitors have obtained clinical approval yet, the proactive development of detection methods for emerging doping agents represents a key aspect of preventive doping research. The presented approach will expand the range of available tests for novel protein therapeutics and can readily be modified to include further target analytes.
Assuntos
Receptores de Activinas Tipo II/sangue , Anticorpos Bloqueadores/sangue , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Análise Química do Sangue/métodos , Proteólise , Tripsina/metabolismo , Receptores de Activinas Tipo II/imunologia , Sequência de Aminoácidos , Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Cromatografia de Afinidade , Cromatografia Líquida , Humanos , Masculino , Espectrometria de MassasRESUMO
The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/imunologia , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Imunização , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Camundongos Transgênicos , Plasmodium falciparum/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/químicaRESUMO
Background: Human norovirus is a significant public health burden, with >30 genotypes causing endemic levels of disease and strains from the GII.4 genotype causing serial pandemics as the virus evolves new ligand binding and antigenicity features. During 2014-2015, genotype GII.17 cluster IIIb strains emerged as the leading cause of norovirus infection in select global locations. Comparison of capsid sequences indicates that GII.17 is evolving at previously defined GII.4 antibody epitopes. Methods: Antigenicity of virus-like particles (VLPs) representative of clusters I, II, and IIIb GII.17 strains were compared by a surrogate neutralization assay based on antibody blockade of ligand binding. Results: Sera from mice immunized with a single GII.17 VLP identified antigenic shifts between each cluster of GII.17 strains. Ligand binding of GII.17 cluster IIIb VLP was blocked only by antisera from mice immunized with cluster IIIb VLPs. Exchange of residues 393-396 from GII.17.2015 into GII.17.1978 ablated ligand binding and altered antigenicity, defining an important varying epitope in GII.17. Conclusions: The capsid sequence changes in GII.17 strains result in loss of blockade antibody binding, indicating that viral evolution, specifically at residues 393-396, may have contributed to the emergence of cluster IIIb strains and the persistence of GII.17 in human populations.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Norovirus/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/química , Anticorpos Antivirais/química , Variação Antigênica , Infecções por Caliciviridae/epidemiologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Variação Genética , Cobaias , Humanos , Imunização , Camundongos , Modelos Moleculares , Norovirus/classificação , Norovirus/genética , Norovirus/ultraestrutura , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.
Assuntos
Anticorpos Bloqueadores/isolamento & purificação , Anticorpos Bloqueadores/metabolismo , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/química , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Xenoenxertos , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos Nus , Transplante de Neoplasias , Ligação Proteica , Conformação Proteica , Proto-Oncogene Mas , Resultado do TratamentoRESUMO
The emergence of multi-drug resistant Neisseria gonorrhoeae has generated an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study we investigate the potential of targeting the surface exposed nitrite reductase, AniA, to block activity by producing functional blocking antibodies. AniA activity is essential for anaerobic growth and biofilm formation of N. gonorrhoeae and functional blocking antibodies may prevent colonisation and disease. Seven peptides covering regions adjacent to the active site were designed based on the AniA structure. Six of the seven peptide conjugates generated immune responses. Peptide 7, GALGQLKVEGAEN, was able to elicit antibodies capable of blocking AniA activity. Antiserum raised against the peptide 7 conjugate detected AniA in 20 N. gonorrhoeae clinical isolates. Recombinant AniA protein antigens were also assessed in this study and generated high-titre, functional blocking antibody responses. Peptide 7 conjugates or truncated recombinant AniA antigens have potential for inclusion in a vaccine against N. gonorrhoeae.
Assuntos
Anticorpos Bloqueadores/imunologia , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Neisseria gonorrhoeae/enzimologia , Peptídeos/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/química , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Domínio Catalítico , Feminino , Imunização , Camundongos , Neisseria gonorrhoeae/imunologia , Peptídeos/agonistas , Peptídeos/síntese química , Coelhos , Proteínas Recombinantes/imunologiaRESUMO
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
Assuntos
Receptor PAR-2/química , Receptor PAR-2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Álcoois Benzílicos/química , Álcoois Benzílicos/farmacologia , Cristalografia por Raios X , Humanos , Imidazóis/química , Imidazóis/farmacologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Cinética , Ligantes , Modelos Moleculares , Receptor PAR-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Conformação Proteica , Ressonância de Plasmônio de Superfície , Difração de Raios XAssuntos
Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Imunidade , Receptor de Morte Celular Programada 1/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Humanos , Ligantes , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Relação Estrutura-AtividadeRESUMO
The invasion-promoting MT1-MMP is a cell surface-associated collagenase with a plethora of critical cellular functions. There is a consensus that MT1-MMP is a key protease in aberrant pericellular proteolysis in migrating cancer cells and, accordingly, a promising drug target. Because of high homology in the MMP family and a limited success in the design of selective small-molecule inhibitors, it became evident that the inhibitor specificity is required for selective and successful MT1-MMP therapies. Using the human Fab antibody library (over 1.25×109 individual variants) that exhibited the extended, 23-27 residue long, VH CDR-H3 segments, we isolated a panel of the inhibitory antibody fragments, from which the 3A2 Fab outperformed others as a specific and potent, low nanomolar range, inhibitor of MT1-MMP. Here, we report the in-depth characterization of the 3A2 antibody. Our multiple in vitro and cell-based tests and assays, and extensive structural modeling of the antibody/protease interactions suggest that the antibody epitope involves the residues proximal to the protease catalytic site and that, in contrast with tissue inhibitor-2 of MMPs (TIMP-2), the 3A2 Fab inactivates the protease functionality by binding to the catalytic domain outside the active site cavity. In agreement with the studies in metastasis by others, our animal studies in acute pulmonary melanoma metastasis support a key role of MT1-MMP in metastatic process. Conversely, the selective anti-MT1-MMP monotherapy significantly alleviated melanoma metastatic burden. It is likely that further affinity maturation of the 3A2 Fab will result in the lead inhibitor and a proof-of-concept for MT1-MMP targeting in metastatic cancers.
Assuntos
Anticorpos Bloqueadores/farmacologia , Antineoplásicos Imunológicos/farmacologia , Metaloproteinase 14 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Anticorpos Bloqueadores/química , Antineoplásicos Imunológicos/química , Ligação Competitiva , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Feminino , Xenoenxertos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Inibidores de Metaloproteinases de Matriz/química , Camundongos , Modelos Moleculares , Conformação Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/tratamento farmacológico , Ligação Proteica , Proteólise , Proteínas Recombinantes/metabolismoRESUMO
Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the ß-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Integrinas/imunologia , Subunidades Proteicas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Sequência Conservada , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/química , Humanos , Integrinas/química , Integrinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Alinhamento de SequênciaRESUMO
In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of â¼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteína Amiloide A Sérica/imunologia , Adalimumab , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Afinidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Receptor gp130 de Citocina/imunologia , Receptor gp130 de Citocina/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/prevenção & controle , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Interleucina-6/metabolismo , Subunidade alfa de Receptor de Interleucina-6/imunologia , Subunidade alfa de Receptor de Interleucina-6/metabolismo , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Proteína Amiloide A Sérica/antagonistas & inibidores , Proteína Amiloide A Sérica/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN). Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite.