RESUMO
Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.
Assuntos
Processamento Alternativo , Lectinas Tipo C/genética , Receptores de Superfície Celular/genética , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células Jurkat , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK/química , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.
Assuntos
Anticorpos Monoclonais/farmacologia , Antirreumáticos/farmacologia , Artrite Reumatoide/etiologia , Desenvolvimento de Medicamentos , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Imuno-Histoquímica , Macaca fascicularis , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismoRESUMO
Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Inflamação/tratamento farmacológico , Proteínas de Membrana/genética , Receptores de Complemento/genética , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Humanos , Inflamação/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a , Receptores de Complemento/metabolismoRESUMO
Inhibition of cytochrome P450 (CYP) enzymes is unwanted because of the risk of severe side effects due to drug-drug interactions. We present two in silico Gaussian kernel weighted k-nearest neighbor models based on extended connectivity fingerprints that classify CYP2D6 and CYP3A4 inhibition. Data used for modeling consisted of diverse sets of 1153 and 1382 drug candidates tested for CYP2D6 and CYP3A4 inhibition in human liver microsomes. For CYP2D6, 82% of the classified test set compounds were predicted to the correct class. For CYP3A4, 88% of the classified compounds were correctly classified. CYP2D6 and CYP3A4 inhibition were additionally classified for an external test set on 14 drugs, and multidimensional scaling plots showed that the drugs in the external test set were in the periphery of the training sets. Furthermore, fragment analyses were performed and structural fragments frequent in CYP2D6 and CYP3A4 inhibitors and noninhibitors are presented.
Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/química , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Inibidores Enzimáticos/química , Modelos Moleculares , Preparações Farmacêuticas/química , Relação Quantitativa Estrutura-Atividade , Análise por Conglomerados , Citocromo P-450 CYP3A , Bases de Dados Factuais , Humanos , Técnicas In Vitro , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologiaRESUMO
Immunogenicity is an important consideration in the licensure of a therapeutic protein because the development of neutralizing anti-drug antibodies (ADAs) can affect both safety and efficacy. Neoantigens introduced by bioengineering of a protein drug are a particular cause for concern. The development of a bioengineered recombinant factor VIIa (rFVIIa) analog was discontinued after phase 3 trials because of the development of ADAs. The unmodified parent molecule (rFVIIa), on the other hand, has been successfully used as a drug for more than two decades with no reports of immunogenicity in congenital hemophilia patients with inhibitors. We used computational and experimental methods to demonstrate that the observed ADAs could have been elicited by neoepitopes in the engineered protein. The human leukocyte antigen type of the patients who developed ADAs is consistent with this hypothesis of a neoepitope-driven immune response, a finding that might have implications for the preclinical screening of therapeutic protein analogs.
Assuntos
Fator VIII/imunologia , Hemofilia A/sangue , Hemofilia A/terapia , Engenharia de Proteínas/métodos , Adolescente , Adulto , Anticorpos Neutralizantes/imunologia , Proliferação de Células , Criança , Estudos Cross-Over , Interpretação Estatística de Dados , Método Duplo-Cego , Epitopos/imunologia , Fator VIIa/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Masculino , Mutação , Proteínas Recombinantes/imunologia , Software , Resultado do Tratamento , Adulto JovemRESUMO
Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-ß superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15-GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/farmacologia , Obesidade/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Ingestão de Alimentos/genética , Metabolismo Energético/genética , Citometria de Fluxo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Ressonância de Plasmônio de Superfície , Redução de Peso/genéticaRESUMO
Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.
Assuntos
Proteínas de Membrana , Biblioteca de Peptídeos , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão , Clonagem Molecular , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
A data set consisting of 712 compounds was used for classification into two classes with respect to membrane permeation in a cell-based assay: (0) apparent permeability (P(app)) below 4 x 10(-6) cm/s and (1) P(app) on 4 x 10(-6) cm/s or higher. Nine molecular descriptors were calculated for each compound and Nearest-Neighbor classification was applied using five neighbors as optimized by full cross-validation. A model based on five descriptors, number of flex bonds, number of hydrogen bond acceptors and donors, and molecular and polar surface area, was selected by variable selection. In an external test set of 112 compounds, 104 compounds were classified and 8 compounds were judged as "unknown". Among the 104 compounds, 16 were misclassified corresponding to a misclassification rate of 15% and no compounds were falsely predicted in the nonpermeable class.
Assuntos
Permeabilidade da Membrana Celular , Absorção Intestinal , Modelos Biológicos , Preparações Farmacêuticas/química , Animais , Linhagem Celular , Difusão , Cães , Humanos , Preparações Farmacêuticas/metabolismo , Relação Quantitativa Estrutura-AtividadeRESUMO
The binding of antigens to antibodies is one of the key events in an immune response against foreign molecules and is a critical element of several biomedical applications including vaccines and immunotherapeutics. For development of such applications, the identification of antibody binding sites (B-cell epitopes) is essential. However experimental epitope mapping is highly cost-intensive and computer-aided methods do in general have moderate performance. One major reason for this moderate performance is an incomplete understanding of what characterizes an epitope. To fill this gap, we here developed a novel framework for comparing and superimposing B-cell epitopes and applied it on a dataset of 107 non-similar antigen:antibody structures extracted from the PDB database. With the presented framework, we were able to describe the general B-cell epitope as a flat, oblong, oval shaped volume consisting of predominantly hydrophobic amino acids in the center flanked by charged residues. The average epitope was found to be made up of â¼15 residues with one linear stretch of 5 or more residues constituting more than half of the epitope size. Furthermore, the epitope area is predominantly constrained to a plane above the antibody tip, in which the epitope is orientated in a -30° to 60° angle relative to the light to heavy chain antibody direction. Contrary to previously findings, we did not find a significant deviation between the amino acid composition in epitopes and the composition of equally exposed parts of the antigen surface. Our results, in combination with previously findings, give a detailed picture of the B-cell epitope that may be used in development of improved B-cell prediction methods.
Assuntos
Complexo Antígeno-Anticorpo/química , Sítios de Ligação de Anticorpos/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Modelos Moleculares , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Dados de Sequência Molecular , Estrutura Quaternária de ProteínaRESUMO
Despite the attractiveness of ion channels as therapeutic targets, there are no examples of monoclonal antibodies directed against ion channels in clinical development. Antibody-mediated inhibition of ion channels could offer a directed, specific therapeutic approach. To investigate the potential of inhibiting ion channel function with an antibody, we focused on Orai1, the pore subunit of the calcium channel responsible for store-operated calcium entry (SOCE) in T cells. Effector T cells are key drivers of autoimmune disease pathogenesis and calcium signaling is essential for T cell activation, proliferation, and cytokine production. We show here the generation of a specific anti-human Orai1 monoclonal antibody (mAb) against an extracellular loop of the plasma membrane-spanning protein. The anti-Orai1 mAb binds native Orai1 on lymphocytes and leads to cellular internalization of the channel. As a result, T cell proliferation, and cytokine production is inhibited in vitro. In vivo, anti-Orai1 mAb is efficacious in a human T cell-mediated graft-versus host disease (GvHD) mouse model. This study demonstrates the feasibility of antibody-mediated inhibition of Orai1 function and, more broadly, reveals the possibility of targeting ion channels with biologics for the treatment of autoimmunity and other diseases.
Assuntos
Anticorpos Monoclonais/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Leucócitos Mononucleares/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/imunologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Hibridomas/imunologia , Transporte de Íons , Células Jurkat , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Camundongos , Dados de Sequência Molecular , Proteína ORAI1 , Cultura Primária de CélulasRESUMO
The structural registration of chemically modified macromolecules is vital for the development of biopharmaceuticals. However, registration and search of such complex molecules has so far posed formidable challenges performance-wise, since today's chemistry-oriented databases do not scale well to macromolecules. As a practical consequence, macromolecules tend to be stored in protein databases with a focus on protein sequence only, and salient chemistry details are therefore lost. This article describes protein format extensions and the use of pseudoatoms for representing natural amino acids in chemical structures to allow high-performance registration and retrieval of large macromolecules. The representations include exact chemical modifications and enable lossless conversion between chemistry and sequence formats. Registration is done in parallel in both sequence and chemistry formats, and users can register and retrieve molecules in either format as they choose, resulting in what we call a BioChemformatics database. Having both sequence and chemistry formats available on-demand allows for the construction of protein SAR tables with mixed sequence and chemistry information. Likewise, searching may combine sequence and chemistry terms and be performed in standard vendor applications like MDL's ISIS/Base or in-house applications using standard SQL queries.