RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Fragmentos Fab de Inmunoglobulinas/química , Fosfopéptidos/química , Proteínas tau/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Sitios de Unión de Anticuerpos , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/metabolismo , Expresión Génica , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Modelos Moleculares , Fosfopéptidos/síntesis química , Fosforilación , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
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.
Asunto(s)
Interleucina-4/química , Interleucina-4/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Repetición de Anquirina , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/farmacologíaRESUMEN
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.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Factores Inmunológicos/química , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Sitios de Unión , Cristalografía por Rayos X , Citocinas/metabolismo , Células HEK293 , Semivida , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Factores Inmunológicos/genética , Factores Inmunológicos/farmacología , Macaca fascicularis , Masculino , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Receptor ErbB-2/inmunología , Receptores de IgG/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Fragmentos Fab de Inmunoglobulinas/química , Animales , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
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.
Asunto(s)
Fibronectinas/química , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Fibronectinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Secuencia de Aminoácidos , Animales , Regiones Determinantes de Complementariedad/química , Cristalografía por Rayos X , Bases de Datos de Proteínas , Humanos , Isomerismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
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.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Estabilidad Proteica , Estructura Terciaria de Proteína , Modelos Moleculares , Tenascina/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Proteolisis , ConejosRESUMEN
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.
Asunto(s)
Interleucina-17/química , Interleucina-17/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Replegamiento Proteico , Dicroismo Circular , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Interleucina-17/aislamiento & purificación , Espectrometría de Masas , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Fragmentos Fab de Inmunoglobulinas/química , Receptor Toll-Like 3/química , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/aislamiento & purificación , Cristalización , Espacio Extracelular/química , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Receptor Toll-Like 3/inmunologíaRESUMEN
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.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Interleucina-13/química , Animales , Complejo Antígeno-Anticuerpo/inmunología , Cristalización , Humanos , Interleucina-13/inmunología , RatonesRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales/metabolismo , Descubrimiento de Drogas/métodos , Proteínas tau/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas tau/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Afinidad de Anticuerpos/fisiología , Tromboplastina/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Regiones Determinantes de Complementariedad/química , Humanos , Ratones , Modelos Moleculares , Ingeniería de Proteínas/métodosRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/metabolismo , Encéfalo/metabolismo , Proteínas tau/inmunología , Animales , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Resonancia por Plasmón de Superficie , Proteínas tau/deficiencia , Proteínas tau/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Complejo Antígeno-Anticuerpo/química , Ligando CD27/química , Fragmentos Fab de Inmunoglobulinas/química , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/genética , Complejo Antígeno-Anticuerpo/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Ligando CD27/genética , Ligando CD27/inmunología , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Células Sf9 , Spodoptera , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
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.
Asunto(s)
Repetición de Anquirina , Anticuerpos/química , Anticuerpos/inmunología , Interleucina-13/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Cristalografía por Rayos X , Humanos , Macaca fascicularis , Modelos Moleculares , Ingeniería de Proteínas , Estructura Secundaria de ProteínaRESUMEN
Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation. MAb 10H10 is unique in that it blocks the signaling pathway and thus inhibits angiogenesis and tumor growth without interfering with coagulation. It was also presumed that mAb 10H10 recognizes the cryptic pool of TF devoid of procoagulant activity. The crystal structure of the 10H10 Fab was determined in the absence and in the presence of the TF extracellular domain (ECD). The structures show that the antibody operates by the key-and-lock mechanism causing no conformational changes in either Fab or TF. The TF:10H10 interface is extensive and includes five segments of TF in both the N-terminal and C-terminal domains of the ECD. Neither the known epitope of FVII, nor the putative epitope of FX overlaps with the 10H10 binding site. The 10H10 epitope points to the likely location of the PAR2 exosite. It is also the hypothetical site of TF interaction with integrins that may play a major role in the encryption-decryption process.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Epítopos/metabolismo , Transducción de Señal , Tromboplastina/química , Tromboplastina/metabolismo , Animales , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ratones , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Ligando CD27/química , Ligando CD27/inmunología , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Semivida , Humanos , Macaca fascicularis , RatonesRESUMEN
To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the 'kinked' conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.
Asunto(s)
Diversidad de Anticuerpos , Regiones Determinantes de Complementariedad/química , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Anticuerpos de Dominio Único/química , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas , Anticuerpos de Dominio Único/genética , SincrotronesRESUMEN
The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.