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Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.
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Anticuerpos Monoclonales , Antígenos , Mapeo Epitopo/métodos , Anticuerpos Monoclonales/química , Antígeno CTLA-4 , EpítoposRESUMEN
Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.
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Trastorno Autístico , Neocórtex , Células Piramidales , Animales , Femenino , Ratones , Embarazo , Trastorno Autístico/genética , Trastorno Autístico/patología , Mutación , Neocórtex/fisiología , Neuronas/fisiología , Células Piramidales/fisiologíaRESUMEN
Sensory processing relies on the correct development of thalamocortical loops. Visual corticothalamic axons (CTAs) invade the dorsolateral geniculate nucleus (dLGN) of the thalamus in early postnatal mice according to a regulated program that includes activity-dependent mechanisms. Spontaneous retinal activity influences the thalamic incursion of CTAs, yet the perinatal thalamus also generates intrinsic patterns of spontaneous activity whose role in modulating afferent connectivity remains unknown. Here, we found that patterned spontaneous activity in the dLGN contributes to proper spatial and temporal innervation of CTAs. Disrupting patterned spontaneous activity in the dLGN delays corticogeniculate innervation under normal conditions and upon eye enucleation. The delayed innervation was evident throughout the first two postnatal weeks but resumes after eye-opening, suggesting that visual experience is necessary for the homeostatic recovery of corticogeniculate innervation.
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Corteza Visual , Ratones , Animales , Tálamo , Cuerpos Geniculados , Axones , Retina , Vías VisualesRESUMEN
BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.
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Neoplasias , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Animales , Anticuerpos Monoclonales/uso terapéutico , Recuento de Células , Epítopos , Humanos , Inmunoterapia , Macaca mulatta , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1RESUMEN
Despite recent advances in transgenic animal models and display technologies, humanization of mouse sequences remains one of the main routes for therapeutic antibody development. Traditionally, humanization is manual, laborious, and requires expert knowledge. Although automation efforts are advancing, existing methods are either demonstrated on a small scale or are entirely proprietary. To predict the immunogenicity risk, the human-likeness of sequences can be evaluated using existing humanness scores, but these lack diversity, granularity or interpretability. Meanwhile, immune repertoire sequencing has generated rich antibody libraries such as the Observed Antibody Space (OAS) that offer augmented diversity not yet exploited for antibody engineering. Here we present BioPhi, an open-source platform featuring novel methods for humanization (Sapiens) and humanness evaluation (OASis). Sapiens is a deep learning humanization method trained on the OAS using language modeling. Based on an in silico humanization benchmark of 177 antibodies, Sapiens produced sequences at scale while achieving results comparable to that of human experts. OASis is a granular, interpretable and diverse humanness score based on 9-mer peptide search in the OAS. OASis separated human and non-human sequences with high accuracy, and correlated with clinical immunogenicity. BioPhi thus offers an antibody design interface with automated methods that capture the richness of natural antibody repertoires to produce therapeutics with desired properties and accelerate antibody discovery campaigns. The BioPhi platform is accessible at https://biophi.dichlab.org and https://github.com/Merck/BioPhi.
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Aprendizaje Profundo , Animales , Anticuerpos , RatonesRESUMEN
Human/humanized IgG4 antibodies have reduced effector function relative to IgG1 antibodies, which is desirable for certain therapeutic purposes. However, the developability and biophysical properties for IgG4 antibodies are not well understood. This work focuses on the head-to-head comparison of key biophysical properties, such as self-interaction and viscosity, for 14 human/humanized, and chimeric IgG1 and IgG4 S228P monoclonal antibody pairs that contain the identical variable regions. Experimental measurements showed that the IgG4 S228P antibodies have similar or higher self-interaction and viscosity than that of IgG1 antibodies in 20 mM sodium acetate, pH 5.5. We report sequence and structural drivers for the increased viscosity and self-interaction detected in IgG4 S228P antibodies through a combination of experimental data and computational models. Further, we applied and extended a previously established computational model for IgG1 antibodies to predict the self-interaction and viscosity behavior for each antibody pair, providing insight into the structural characteristics and differences of these two isotypes. Interestingly, we observed that the IgG4 S228P swapped variants, where the CH3 domain was swapped for that of an IgG1, showed reduced self-interaction behavior. These domain swapped IgG4 S228P molecules also showed reduced viscosity from experiment and coarse-grained simulations. We also observed that experimental diffusion interaction parameter (kD) values have a high correlation with computational diffusivity prediction for both IgG1 and IgG4 S228P isotypes.Abbreviations: AHc, constant region Hamaker constant; AHv, variable region Hamaker constant; CDRs, Complementarity-determining regions; CG, Coarse-grained model; CH1, Constant heavy chain 1; CH2 Constant heavy chain 2; CH3 Constant heavy chain 3; chgCH3 Effective charge on the CH3 region; CL Constant light chain; cP, Centipoise; DLS, Dynamic light scattering; Fab, Fragment antigen-binding; Fc, Fragment crystallizable; Fv, Variable domaing; (r) Radial distribution function; H1 CDR1 of Heavy Chain; H2 CDR2 of Heavy Chain; H3 CDR3 of Heavy Chain; HVI, High viscosity index; IgG1 human immunoglobulin of IgG1 subclass; IgG4 human immunoglobulin of IgG4 subclass; kD, Diffusion interaction parameter; L1 CDR1 of Light Chain; L2 CDR2 of Light Chain; L3 CDR3 of Light Chain; mAb, Monoclonal antibody; MD, Molecular dynamics; PPI Protein-protein interactions; SCM, Spatial charge map; UP-SEC, Ultra-high-performance size-exclusion chromatography; VH, Variable domain of Heavy Chain; VL, Variable domain of Light Chain.
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Anticuerpos Monoclonales , Inmunoglobulina G , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Regiones Determinantes de Complementariedad/química , Humanos , Inmunoglobulina G/química , ViscosidadRESUMEN
Developing sensory circuits exhibit different patterns of spontaneous activity, patterns that are related to the construction and refinement of functional networks. During the development of different sensory modalities, spontaneous activity originates in the immature peripheral sensory structures and in the higher-order central structures, such as the thalamus and cortex. Certainly, the perinatal thalamus exhibits spontaneous calcium waves, a pattern of activity that is fundamental for the formation of sensory maps and for circuit plasticity. Here, we review our current understanding of the maturation of early (including embryonic) patterns of spontaneous activity and their influence on the assembly of thalamic and cortical sensory networks. Overall, the data currently available suggest similarities between the developmental trajectory of brain activity in experimental models and humans, which in the future may help to improve the early diagnosis of developmental disorders.
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Potenciales de Acción/fisiología , Corteza Cerebral/fisiología , Plasticidad Neuronal/fisiología , Corteza Somatosensorial/fisiología , Animales , Humanos , Neuronas/fisiología , Lóbulo Parietal/fisiologíaRESUMEN
Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.
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Arginasa/genética , Arginasa/metabolismo , Arginina/química , Sitios de Unión , Microscopía por Crioelectrón , Ornitina/química , Unión Proteica , Especificidad por SustratoRESUMEN
Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.
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Neuronas , Receptor de Adenosina A2A , Animales , Axones , Movimiento Celular/fisiología , Interneuronas , Ratones , Neuronas/fisiología , Células Piramidales/fisiología , Receptor de Adenosina A2A/genéticaRESUMEN
Neural cell diversity is essential to endow distinct brain regions with specific functions. During development, progenitors within these regions are characterized by specific gene expression programs, contributing to the generation of diversity in postmitotic neurons and astrocytes. While the region-specific molecular diversity of neurons and astrocytes is increasingly understood, whether these cells share region-specific programs remains unknown. Here, we show that in the neocortex and thalamus, neurons and astrocytes express shared region-specific transcriptional and epigenetic signatures. These signatures not only distinguish cells across these two brain regions but are also detected across substructures within regions, such as distinct thalamic nuclei, where clonal analysis reveals the existence of common nucleus-specific progenitors for neurons and astrocytes. Consistent with their shared molecular signature, regional specificity is maintained following astrocyte-to-neuron reprogramming. A detailed understanding of these regional-specific signatures may thus inform strategies for future cell-based brain repair.
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Astrocitos , Neocórtex , Astrocitos/metabolismo , Epigenómica , Neuronas/fisiología , TálamoRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2019.01151.].
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Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.
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Anticuerpos Monoclonales , Descubrimiento de Drogas/métodos , Flujo de Trabajo , Humanos , Ingeniería de Proteínas/métodosRESUMEN
Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.
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Assessment of the factors that regulate antibody exposure-response relationships in the relevant animal models is critical for the design of successful translational strategies from discovery to the clinic. Depending on the specific clinical indication, preclinical development paradigms may require that the efficacy or dosing-related attributes for the existing antibody be assessed in various species when cross-reactivity of the lead antibody to the intended species is justified. Additionally, with the success of monoclonal antibodies for management of various human conditions, a parallel interest in therapeutic use of these novel modalities in various veterinary species has followed. The protective role of neonatal Fc receptor (FcRn) in regulation of IgG homeostasis and clearance is now well recognized and the "nonspecific clearance" of antibodies through bone marrow-derived phagocytic and vascular endothelial cells (via lysosomal processes) is modulated by interactions with FcRn receptors. In this study, we have attempted to examine the PK properties of human IgG antibodies in dog and monkey. These studies establish a translational framework for evaluation of IgG antibody PK properties across species.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Inmunoglobulina G/farmacología , Administración Intravenosa , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Macaca fascicularis , Macaca mulatta , Ratones , Modelos Animales , Virus Sincitiales Respiratorios/inmunología , Especificidad de la EspecieRESUMEN
The development of cortical maps requires the balanced interaction between genetically determined programs and input/activity-dependent signals generated spontaneously or triggered from the environment. The somatosensory pathway of mice provides an excellent scenario to study cortical map development because of its highly organized cytoarchitecture, known as the barrel field. This precise organization makes evident even small alterations in the cortical map layout. In this review, we will specially focus on the thalamic factors that control barrel field development. We will summarize the role of thalamic input integration and identity, neurotransmission and spontaneous activity in cortical map formation and early cross-modal plasticity.
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Regulación de la Expresión Génica/fisiología , Plasticidad Neuronal/fisiología , Corteza Somatosensorial/anatomía & histología , Corteza Somatosensorial/crecimiento & desarrollo , Tálamo/fisiología , Animales , Ratones , Tálamo/metabolismoRESUMEN
The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorß upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.
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Núcleos Talámicos Ventrales/anatomía & histología , Núcleos Talámicos Ventrales/embriología , Animales , Calcio/metabolismo , Femenino , Uniones Comunicantes/metabolismo , Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Plasticidad Neuronal , Receptores Nucleares Huérfanos/genética , Embarazo , Corteza Somatosensorial/fisiología , Núcleos Talámicos Ventrales/metabolismo , Núcleos Talámicos Ventrales/fisiología , Visión OcularRESUMEN
The thalamus is a central brain structure with topographically ordered long-range axonal projections that convey sensory information to the cortex via distinct nuclei. Although there is an increasing knowledge about genes important for thalamocortical (TC) development, the identification of genetic landmarks of the distinct thalamic nuclei during the embryonic development has not been addressed systematically. Indeed, a more comprehensive understanding of how the axons from the individual nuclei find their way and connect to their corresponding cortical area is called for. Here, we used a genetic dual labeling strategy in mice to purify distinct principal sensory thalamic neurons. Subsequent genome-wide transcriptome profiling revealed genes specifically expressed in each nucleus during embryonic development. Analysis of regulatory regions of the identified genes revealed key transcription factors and networks that likely underlie the specification of individual sensory-modality TC connections. Finally, the importance of correct axon targeting for the specific sensory-modality population transcriptome was evidenced in a Sema6A mutant, in which visual TC axons are derailed at embryonic life. In sum, our data determined the developmental transcriptional profile of the TC neurons that will eventually support sensory processing.
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Corteza Cerebral/citología , Corteza Cerebral/embriología , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Núcleos Talámicos/citología , Núcleos Talámicos/embriología , Animales , Axones/metabolismo , Corteza Cerebral/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones Transgénicos , Mutación , Vías Nerviosas/citología , Vías Nerviosas/embriología , Vías Nerviosas/metabolismo , Semaforinas/deficiencia , Semaforinas/genética , Núcleos Talámicos/metabolismo , TranscriptomaRESUMEN
Controlling the axon growth rate is fundamental when establishing brain connections. Using the thalamocortical system as a model, we previously showed that spontaneous calcium activity influences the growth rate of thalamocortical axons by regulating the transcription of Robo1 through an NF-κB-binding site in its promoter. Robo1 acts as a brake on the growth of thalamocortical axons in vivo. Here, we have identified the Netrin-1 receptor DCC as an accelerator for thalamic axon growth. Dcc transcription is regulated by spontaneous calcium activity in thalamocortical neurons and activating DCC signaling restores normal axon growth in electrically silenced neurons. Moreover, we identified an AP-1-binding site in the Dcc promoter that is crucial for the activity-dependent regulation of this gene. In summary, we have identified the Dcc gene as a novel downstream target of spontaneous calcium activity involved in axon growth. Together with our previous data, we demonstrate a mechanism to control axon growth that relies on the activity-dependent regulation of two functionally opposed receptors, Robo1 and DCC. These two proteins establish a tight and efficient means to regulate activity-guided axon growth in order to correctly establish neuronal connections during development.
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Axones/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Tálamo/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Axones/ultraestructura , Sitios de Unión , Calcio/metabolismo , Células Cultivadas , Receptor DCC , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Conos de Crecimiento/fisiología , Ratones , FN-kappa B/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Netrina-1 , Neuronas/fisiología , Regiones Promotoras Genéticas , Receptores de Superficie Celular/química , Transducción de Señal , Tálamo/citología , Tálamo/embriología , Proteínas Supresoras de Tumor/químicaRESUMEN
We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.