RESUMEN
Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).
Asunto(s)
Alanina/metabolismo , Anticuerpos Monoclonales/metabolismo , Mapeo Epitopo/métodos , Epítopos/metabolismo , Interleucina-23/metabolismo , Reacciones Antígeno-Anticuerpo , Clonación Molecular , Medición de Intercambio de Deuterio , Fragmentos Fab de Inmunoglobulinas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Mutagénesis , Oxidación-Reducción , Unión ProteicaRESUMEN
BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate. Based on this analysis, the EC10 value (0.32 nM) from the human MLR assay and the human plasma volume (0.04 l/kg) were employed to calculate the MABEL (0.01 mg) of BMS-931699 in humans, with a CD28 RO predicted to be ≤10%. The estimated MABEL dose was threefold higher than the value derived from the binding constant and twofold less than the MABEL converted from animal efficacy studies based on the body surface area. Furthermore, it was 2900-fold lower than the human equivalent dose derived from the no observed adverse effect level in monkeys (15 mg/kg/week for 5 doses, intravenous dosing) with a 10-fold safety factor applied. Therefore, the MABEL dose represented a sound approach to mitigate any potential risk in targeting CD28 and was successfully used as the first-in-human starting dose for BMS-931699.
Asunto(s)
Anticuerpos/farmacología , Antígenos CD28/antagonistas & inhibidores , Polietilenglicoles/farmacología , Polietilenglicoles/farmacocinética , Algoritmos , Animales , Superficie Corporal , Relación Dosis-Respuesta a Droga , Femenino , Hemocianinas/antagonistas & inhibidores , Humanos , Prueba de Cultivo Mixto de Linfocitos , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Pruebas de Sensibilidad Microbiana , Monocitos/efectos de los fármacosRESUMEN
Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance-possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.
Asunto(s)
Imagenología Tridimensional/métodos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Desplegamiento Proteico , Análisis Espectral/métodos , Humanos , Cinética , Unión Proteica , Conformación ProteicaRESUMEN
Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.
Asunto(s)
Anticuerpos/química , Bioensayo/métodos , Polietilenglicoles/química , Semivida , Cinética , Microscopía de Fuerza Atómica/métodos , Unión Proteica/efectos de los fármacos , Proteínas/química , Resonancia por Plasmón de Superficie/métodosRESUMEN
The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3â Å resolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.
Asunto(s)
Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/química , Secuencia de Aminoácidos , Calorimetría , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/aislamiento & purificación , Ligando Inductor de Apoptosis Relacionado con TNF/aislamiento & purificación , TermodinámicaRESUMEN
Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked. However, 2B4 up-regulation was not observed in animals treated with CTLA-4 Ig (abatacept) or CD28 blockade in the presence of anti-CTLA-4 mAb. 2B4 up-regulation after CD28 blockade was functionally significant, as the inhibitory impact of CD28 blockade was diminished when antigen-specific CD8(+) T cells were deficient in 2B4. In contrast, 2B4 deficiency had no effect on CD8(+) T cell responses during unmodified rejection or in the presence of CTLA-4 Ig. We conclude that blockade of CD28 signals in the presence of preserved CTLA-4 signals results in the unique up-regulation of 2B4 on primary CD8(+) effectors, and that this 2B4 expression plays a critical functional role in controlling antigen-specific CD8(+) T cell responses.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD28/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Receptores Inmunológicos/biosíntesis , Abatacept , Aloinjertos , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD/genética , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Supervivencia de Injerto/inmunología , Inmunoconjugados/administración & dosificación , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Transducción de Señal/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Trasplante de Piel , Regulación hacia ArribaRESUMEN
It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.
Asunto(s)
Apolipoproteína A-II/química , Lipoproteínas HDL/química , Lipoproteínas/química , Apolipoproteína A-II/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Estructura Secundaria de Proteína , Espectrofotometría Infrarroja , Relación Estructura-ActividadRESUMEN
The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/química , Glicoproteínas de Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores del Factor de Necrosis Tumoral/química , Animales , Afinidad de Anticuerpos , Bioensayo , Diferenciación Celular , Membrana Celular/metabolismo , Dicroismo Circular , Citocinas/metabolismo , Dimerización , Disulfuros/química , Escherichia coli/metabolismo , Vectores Genéticos , Glicoproteínas/metabolismo , Heparina/química , Humanos , Insectos , Cinética , Ligandos , Modelos Químicos , Mutación , Osteoclastos/metabolismo , Osteoprotegerina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Resonancia por Plasmón de Superficie , Factores de Tiempo , Virus/genéticaRESUMEN
Apolipoprotein E (apoE) is a key regulator of cholesterol homeostasis. Human apoE has three common isoforms, each with different risk implications for cardiovascular and neurodegenerative disease. Neither the structure of lipoprotein E particles nor the structural consequences of the isoform differences are known. In this investigation, synthetic lipoprotein particles were prepared by complexing phospholipids with full-length apoE isoforms, or with truncated N-terminal and C-terminal domains of apoE. These particles were examined with calorimetry, electron microscopy, circular dichroism spectroscopy, and internal reflection infrared spectroscopy. Results indicate that particles made with the three full-length apoE isoforms are discoidal in shape, and structurally indistinguishable. Thus, differences in their pathological consequences are not due to gross differences in particle structure. Although apoE is predominantly helical, and the axes of the helices are parallel to the flat surfaces of the particles, the orientational order of lipid acyl chains is low and inconsistent with the belt model of lipoprotein A-I structure. Instead, the data suggest that there are at least two different types of apoE-lipid interactions within lipoprotein E particles. One type occurs between apoE helices and the edge of the lipid bilayer as in the belt model, while a second type involves apoE helices that situate in the plane of the membrane and disturb acyl chain order. These interactions allow LpE particles to form with different protein/lipid ratios, and they account for the structure of LpE particles made with only the truncated domains.
Asunto(s)
Apolipoproteínas E/química , Apolipoproteínas E/ultraestructura , Cristalografía por Rayos X , Microscopía Electrónica de Rastreo , Modelos Moleculares , Fragmentos de Péptidos/química , Fosfolípidos , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.