RESUMO
This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.
Assuntos
Proteínas de Bactérias/química , Domínio de Fibronectina Tipo III , Cloreto de Sódio/química , Thermoanaerobacter/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Alta , Simulação de Dinâmica Molecular , Domínios Proteicos , Estabilidade Proteica , Thermoanaerobacter/genéticaRESUMO
Microtubule-associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF-tau). AT8 is a PHF-tau-specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30-fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF-tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202-209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR-L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody.
Assuntos
Anticorpos Monoclonais/química , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Fosfopeptídeos/química , Proteínas tau/química , Sequência de Aminoácidos , Anticorpos Monoclonais/biossíntese , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Modelos Moleculares , Fosfopeptídeos/síntese química , Fosforilação , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
The crystal structure of DARPin 44C12V5 that neutralizes IL-4 signaling has been determined alone and bound to human IL-4. A significant conformational change occurs in the IL-4 upon DARPin binding. The DARPin binds to the face of IL-4 formed by the A and C α-helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL-4 include a reorientation of the C-helix Trp91 side chain and repositioning of CD-loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL-4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical "hotspot." The structures also reveal how the DARPin neutralizes IL-4 signaling. Comparing the IL-4:DARPin complex structure with the structures of IL-4 bound to its receptors (Hage et al., Cell 1999; 97, 271-281; La Porte et al., Cell 2008, 132, 259-272), it is found that the DARPin binds to the same IL-4 face that interacts with the junction of the D1 and D2 domains of the IL-4Rα receptors. Signaling is blocked since IL-4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL-13α1 or the γc receptor.
Assuntos
Interleucina-4/química , Interleucina-4/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Repetição de Anquirina , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/farmacologiaRESUMO
The Fc variant of IgG2, designated as IgG2σ, was engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement protein and consequently, immune effector functions. IgG2σ was compared to other previously well-characterized Fc 'muted' variants, including aglycosylated IgG1, IgG2m4 (H268Q/V309L/A330S/P331S, changes to IgG4), and IgG4 ProAlaAla (S228P/L234A/L235A) in its capacity to bind FcγRs and activate various immune-stimulatory responses. In contrast to the previously characterized muted Fc variants, which retain selective FcγR binding and effector functions, IgG2σ shows no detectable binding to the Fcγ receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2σ shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2σ in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcγRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcγRs. Inspection of the structure also indicated significant changes to the conformations of D270 and P329 in the CH2 domain that could negatively impact C1q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2σ suggest that it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fc-fusion platforms.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Fatores Imunológicos/química , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Sítios de Ligação , Cristalografia por Raios X , Citocinas/metabolismo , Células HEK293 , Meia-Vida , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Macaca fascicularis , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Receptor ErbB-2/imunologia , Receptores de IgG/químicaRESUMO
Three Fab structures used as targets in the Antibody Modeling Assessment presented a challenge for modeling CDR-L3 due to deviations from the most typical canonical structure. In all three antibodies CDR-L3 has eight residues, which is one residue shorter than usual, and has a conformation that is rarely observed in crystal structures. We analyzed the sequence and structural determinants of this conformation and found that the "short" CDR-L3 is remarkably rigid and retains the conformation in the interactions with antigens and neighboring CDRs.
Assuntos
Anticorpos Monoclonais/química , Regiões Determinantes de Complementaridade/química , Fragmentos Fab das Imunoglobulinas/química , Animais , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , RatosRESUMO
The crystal structures of six different fibronectin Type III consensus-derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-terminal ß-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the ß-strand-swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non-glycine residue at position 77 and insertions of <4 residues may destabilize the ß-turn in the FG loop promoting ß-strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect ß-bridges in the FG regions that link the swapped dimers. The Tencon ß-swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain-swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties.
Assuntos
Fibronectinas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Cristalografia por Raios X , Fibronectinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
To assess the state-of-the-art in antibody structure modeling, a blinded study was conducted. Eleven unpublished Fab crystal structures were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. In the first round, each participant submitted three non-ranked complete Fv models for each target. In the second round, CDR-H3 modeling was performed in the context of the correct environment provided by the crystal structures with CDR-H3 removed. In this report we describe the reference structures and present our assessment of the models. Some of the essential sources of errors in the predictions were traced to the selection of the structure template, both in terms of the CDR canonical structures and VL/VH packing. On top of this, the errors present in the Protein Data Bank structures were sometimes propagated in the current models, which emphasized the need for the curated structural database devoid of errors. Modeling non-canonical structures, including CDR-H3, remains the biggest challenge for antibody structure prediction.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Regiões Determinantes de Complementaridade/química , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The crystal structure of an N-terminal ß-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal ß-strand swapping, leading to the formation of a stable compact dimer.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica , Estrutura Terciária de Proteína , Modelos Moleculares , Tenascina/químicaRESUMO
The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Proteólise , CoelhosRESUMO
Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Humanos , Staphylococcus aureus , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/microbiologia , Anticorpos Monoclonais/uso terapêutico , Fagócitos/metabolismo , Leucocidinas/metabolismo , Leucocidinas/uso terapêuticoRESUMO
Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.
Assuntos
Alelos , Linfócitos T CD8-Positivos/imunologia , COVID-19 , Antígenos de Histocompatibilidade Classe I/imunologia , Células T de Memória/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , SARS-CoV-2/imunologia , COVID-19/genética , COVID-19/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , SARS-CoV-2/genéticaRESUMO
A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.
Assuntos
Interleucina-17/química , Interleucina-17/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Redobramento de Proteína , Dicroísmo Circular , Escherichia coli/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Interleucina-17/isolamento & purificação , Espectrometria de Massas , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
C836 is a neutralizing monoclonal antibody to human interleukin IL-13 generated by mouse immunization. The crystal structure of the C836 Fab was determined at 2.5 Å resolution and compared with the IL-13-bound form determined previously. This comparison indicates an induced-fit mechanism of antigen recognition through rigid-body rotation of the V(L) and V(H) domains. The magnitude of this rearrangement is one of the largest observed for antibody-protein interactions.
Assuntos
Antígenos/química , Região Variável de Imunoglobulina/química , Animais , Antígenos/imunologia , Cristalografia por Raios X , Região Variável de Imunoglobulina/imunologia , Camundongos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization.
Assuntos
Complexo Antígeno-Anticorpo/química , Fragmentos Fab das Imunoglobulinas/química , Receptor 3 Toll-Like/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/isolamento & purificação , Cristalização , Espaço Extracelular/química , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Receptor 3 Toll-Like/imunologiaRESUMO
The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate.
Assuntos
Complexo Antígeno-Anticorpo/química , Interleucina-13/química , Animais , Complexo Antígeno-Anticorpo/imunologia , Cristalização , Humanos , Interleucina-13/imunologia , CamundongosRESUMO
BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais/metabolismo , Descoberta de Drogas/métodos , Proteínas tau/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Proteínas tau/químicaRESUMO
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.
Assuntos
Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/fisiologia , Tromboplastina/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Regiões Determinantes de Complementaridade/química , Humanos , Camundongos , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.
Assuntos
Doença de Alzheimer/patologia , Anticorpos Monoclonais/metabolismo , Encéfalo/metabolismo , Proteínas tau/imunologia , Animais , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Imunização Passiva , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Ressonância de Plasmônio de Superfície , Proteínas tau/deficiência , Proteínas tau/genéticaRESUMO
CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex with the Fab fragment of mAb 2177 was determined at 1.8â Å resolution. CD27 exhibits the assembly of cysteine-rich domains characteristic of the TNF receptor superfamily. The structure reveals a unique binding site of mAb 2177 at the edge of the receptor molecule, which allows the mAb to sterically block the cell-bound form of CD70 from reaching CD27 while leaving the ligand epitope clear. This mode of action suggests a potential dual use of mAb 2177 either as an antagonist or as an agonist.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Complexo Antígeno-Anticorpo/química , Ligante CD27/química , Fragmentos Fab das Imunoglobulinas/química , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/química , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Complexo Antígeno-Anticorpo/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Ligante CD27/genética , Ligante CD27/imunologia , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Células Sf9 , Spodoptera , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Designed ankyrin repeat proteins (DARPin®) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution. The DARPin competes for the same epitope as the IL-13 receptor chain 13Rα1 but does not interfere with the binding of the other receptor chain, IL-4Rα. Analysis of multiple copies of the DARPin molecule in the crystal indicates the conformational instability in the N-terminal cap that was predicted from molecular dynamics simulations. Comparison of the DARPin structures in the free state and in complex with IL-13 reveals a concerted movement of the ankyrin repeats upon binding resulted in the opening of the binding site. The induced-fit mode of binding employed by DARPin 6G9 is very unusual for DARPins since they were designed as particularly stable and rigid molecules. This finding shows that DARPins can operate by various binding mechanisms and suggests that some flexibility in the scaffold may be an advantage.