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Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
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Aminoaciltransferases , Linfócitos T CD8-Positivos , Quimiocinas , Neoplasias , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Linfócitos T CD8-Positivos/patologia , Quimiocinas/metabolismo , Imunoterapia , Infiltração Leucêmica , Camundongos , Camundongos Knockout , Monócitos , Neoplasias/imunologiaRESUMO
Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
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Doença de Alzheimer , Glicoproteínas de Membrana , Microglia , Esclerose Múltipla , Receptores Imunológicos , Animais , Receptores Imunológicos/agonistas , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Glicoproteínas de Membrana/agonistas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Feminino , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anticorpos/farmacologia , Humanos , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMO
INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
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Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.
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Doenças Neurodegenerativas , Doença de Parkinson , Sinucleinopatias , Camundongos , Animais , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Doenças Neurodegenerativas/metabolismo , Sinucleinopatias/patologia , Neurônios Dopaminérgicos/metabolismoRESUMO
The surface envelope glycoproteins of nonprimate lentiviruses and betaretroviruses share sequence similarity with the inner proximal domain ß-sandwich of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein that faces the transmembrane glycoprotein as well as patterns of cysteine and glycosylation site distribution that points to a similar two-domain organization in at least some lentiviruses. Here, high-reliability models of the surface glycoproteins obtained with the AlphaFold algorithm are presented for the gp135 glycoprotein of the small ruminant caprine arthritis-encephalitis (CAEV) and visna lentiviruses and the betaretroviruses Jaagsiekte sheep retrovirus (JSRV), mouse mammary tumor virus (MMTV), and consensus human endogenous retrovirus type K (HERV-K). The models confirm and extend the inner domain structural conservation in these viruses and identify two outer domains with a putative receptor binding site in the CAEV and visna virus gp135. The location of that site is consistent with patterns of sequence conservation and glycosylation site distribution in gp135. In contrast, a single domain is modeled for the JSRV, MMTV, and HERV-K betaretrovirus envelope proteins that is highly conserved structurally in the proximal region and structurally diverse in apical regions likely to interact with cell receptors. The models presented here identify sites in small ruminant lentivirus and betaretrovirus envelope glycoproteins likely to be critical for virus entry and virus neutralization by antibodies and will facilitate their functional and structural characterization. IMPORTANCE Structural information on the surface envelope proteins of lentiviruses and related betaretroviruses is critical to understand mechanisms of virus-host interactions. However, experimental determination of these structures has been challenging, and only the structure of the human immunodeficiency virus type 1 gp120 has been determined. The advent of the AlphaFold artificial intelligence method for structure prediction allows high-quality modeling of the structures of small ruminant lentiviral and betaretroviral surface envelope proteins. The models are consistent with much of the previously described experimental data, show regions likely to interact with receptors, and identify domains that may be involved in mechanisms of antibody neutralization resistance in the small ruminant lentiviruses. The models will allow more precise design of mutants to further determine mechanisms of viral entry and immune evasion in this group of viruses and constructs for structural determination of these surface envelope proteins.
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Algoritmos , Betaretrovirus/química , Produtos do Gene env/química , Lentivirus/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Retrovirus Endógenos/química , Produtos do Gene env/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , RuminantesRESUMO
The retroviral surface envelope protein subunit (SU) mediates receptor binding and triggers membrane fusion by the transmembrane (TM) subunit. SU evolves rapidly under strong selective conditions, resulting in seemingly unrelated SU structures in highly divergent retroviruses. Structural modeling of the SUs of several retroviruses and related endogenous retroviral elements with AlphaFold 2 identifies a TM-proximal SU ß-sandwich structure that has been conserved in the orthoretroviruses for at least 110 million years. The SU of orthoretroviruses diversified by the differential expansion of the ß-sandwich core to form domains involved in virus-host interactions. The ß-sandwich domain is also conserved in the SU equivalent GP1 of Ebola virus although with a significantly different orientation in the trimeric envelope protein structure relative to the ß-sandwich of human immunodeficiency virus type 1 gp120, with significant evidence for divergent rather than convergent evolution. The unified structural view of orthoretroviral SU and filoviral GP1 identifies an ancient, structurally conserved, and evolvable domain underlying the structural diversity of orthoretroviral SU and filoviral GP1. IMPORTANCE The structural relationships of SUs of retroviral groups are obscured by the high rate of sequence change of SU and the deep-time divergence of retroviral lineages. Previous data showed no structural or functional relationships between the SUs of type C gammaretroviruses and lentiviruses. A deeper understanding of structural relationships between the SUs of different retroviral lineages would allow the generalization of critical processes mediated by these proteins in host cell infection. Modeling of SUs with AlphaFold 2 reveals a conserved core domain underlying the structural diversity of orthoretroviral SUs. Definition of the conserved SU structural core allowed the identification of a homologue structure in the SU equivalent GP1 of filoviruses that most likely shares an origin, unifying the SU of orthoretroviruses and GP1 of filoviruses into a single protein family. These findings will allow an understanding of the structural basis for receptor-mediated membrane fusion mechanisms in a broad range of biomedically important retroviruses.
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Ebolavirus , Retrovirus Endógenos , Proteínas do Envelope Viral , Ebolavirus/metabolismo , Retrovirus Endógenos/metabolismo , Produtos do Gene env/química , Produtos do Gene env/metabolismo , Humanos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.
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Peptídeos , Pró-Colágeno , Animais , Espectrometria de Massas , Camundongos , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Proteínas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Proteolytic processing of procollagens is a central step during collagen fibril formation. Bone morphogenic protein 1 (BMP1) is a metalloprotease that plays an important role in the cleavage of carboxy-terminal (COOH-terminal) propeptides from procollagens. Although the removal of propeptides is required to generate mature collagen fibrils, the contribution of BMP1 to this proteolytic process and its action site remain to be fully determined. In this study, using postnatal lung fibroblasts as a model system, we showed that genetic ablation of Bmp1 in primary murine lung fibroblasts abrogated COOH-terminal cleavage from type I procollagen as measured by COOH-terminal propeptide of type I procollagen (CICP) production. We also showed that inhibition of BMP1 by siRNA-mediated knockdown or small-molecule inhibitor reduced the vast majority of CICP production and collagen deposition in primary human lung fibroblasts. Furthermore, we discovered and characterized two antibody inhibitors for BMP1. In both postnatal lung fibroblast and organoid cultures, BMP1 blockade prevented CICP production. Together, these findings reveal a nonredundant role of extracellular BMP1 to process CICP in lung fibroblasts and suggest that development of antibody inhibitors is a viable pharmacological approach to target BMP1 proteinase activity in fibrotic diseases.
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Proteína Morfogenética Óssea 1/metabolismo , Líquido Extracelular/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Proteólise , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Proteína Morfogenética Óssea 1/genética , Células CHO , Cricetinae , Cricetulus , Líquido Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células HEK293 , Humanos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Organoides , Oxidiazóis/farmacologia , Fragmentos de Peptídeos/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Pró-Colágeno/genética , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , CoelhosRESUMO
Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.
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Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/fisiopatologia , Diafragma/fisiopatologia , Junção Neuromuscular/fisiopatologia , Receptores Proteína Tirosina Quinases/agonistas , Esclerose Lateral Amiotrófica/patologia , Animais , Diafragma/patologia , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologia , Junção Neuromuscular/patologia , Superóxido Dismutase-1/genéticaRESUMO
A critical part of the clinical development path for a therapeutic antibody involves evaluating the physical and chemical stability of candidate molecules throughout the manufacturing process. In particular, the risks of chemical liabilities that can impact antigen binding, such as deamidation, oxidation, and isomerization in the antibody CDR sequences, need to be controlled through formulation development or eliminated by replacing the amino acid motif displaying the chemical instability. Commonly, the antibody CDR sequence contains multiple sequence motifs (potential hotspots) for chemical instability. However, only a subset of these motifs results in actual chemical modification, and thus, experimental assessment of the extent of instability is necessary to identify positions for potential sequence engineering. Ideally, this information should be available prior to antibody humanization at the stage of parental rodent antibody identification. Early knowledge of liabilities allows for ranking of clones or the mitigation of liabilities by concurrent engineering with the antibody humanization process instead of time-consuming sequential activities. However, concurrent engineering of chemical liabilities and humanization requires translatability of the chemical modifications from the rodent parental antibody to the humanized. We experimentally compared the stability of all sequence motifs by mass spectrometric peptide mapping between the rodent parental antibody and the final humanized antibody and observed a linear correlation. These results have enabled a streamlined developability assessment process for therapeutic antibodies from lead discovery to clinical development.
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Anticorpos/imunologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Desaminação , Concentração de Íons de Hidrogênio , Isomerismo , Metionina/química , Camundongos , Oxirredução , Espectrometria de Massas em Tandem , Triptofano/químicaRESUMO
The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.
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Região Variável de Imunoglobulina/imunologia , Farmacocinética , Animais , Ensaio de Imunoadsorção Enzimática , Região Variável de Imunoglobulina/química , Macaca fascicularis , Medição de RiscoRESUMO
Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after in vivo administration. Because of the complexity of in vivo samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC-MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC-MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between in vivo samples and samples stressed in vitro at neutral pH were consistent, suggesting that the in vitro stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies in vivo.
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Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Desaminação , Animais , Anticorpos/sangue , Anticorpos/uso terapêutico , Automação , Células CHO , Cromatografia Líquida , Cricetulus , Eritrócitos , Feminino , Citometria de Fluxo , Humanos , Macaca fascicularis , Espectrometria de MassasRESUMO
Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.
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Modelos Moleculares , Conformação Proteica , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Cristalografia , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína/genética , Receptores de Glucagon/antagonistas & inibidoresRESUMO
Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.
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Anticorpos Monoclonais/imunologia , Receptores de Glucagon/química , Receptores de Glucagon/imunologia , Regulação Alostérica , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Espaço Extracelular/metabolismo , Humanos , Masculino , Camundongos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Glucagon/antagonistas & inibidoresRESUMO
Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 mL) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 mL) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.
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Apicomplexan protozoa are intracellular parasites of medical and economic importance. These parasites contain specialized apical complex organelles, including rhoptries, that participate in the process of host cell invasion. Conserved antigens expressed in the rhoptries are rational vaccine targets, but whether conservation of protein structure is a functional requirement for invasion remains unknown. Novel protein structural modeling enables identification of structurally conserved protein families that are not evident by sequence analysis alone. Here we show by AlphaFold2 structural modeling that the rhoptry-associated protein 1 superfamily of the Piroplasmida hemoparasites Babesia and Theileria (pRAP-1) is structurally conserved, with the core conserved region being composed of a globin-like and a 4-helix bundle subdomain. Search for structurally related members of this protein family in other apicomplexan parasites revealed structural homologues of pRAP-1 in several species of Plasmodium, Toxoplasma gondii and other members of the Sarcocystidae family. Based on these structural findings, pRAP-1 is a conserved apical complex protein, but whether these proteins share functional features in different species remains unknown. Identification of widely conserved elements involved in infection in these parasites will enhance our knowledge of invasion mechanisms, and facilitate the design of methods for controlling diseases that affect humans and animals globally.
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Babesia , Parasitos , Plasmodium , Toxoplasma , Humanos , Animais , Interações Hospedeiro-Parasita , Proteínas de Protozoários/metabolismo , Parasitos/metabolismo , Plasmodium/genética , Plasmodium/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Babesia/metabolismo , Organelas/metabolismoRESUMO
Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.
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Doença de Alzheimer , Tauopatias , Doença de Alzheimer/tratamento farmacológico , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Imunização Passiva , Camundongos , Camundongos Transgênicos , Tauopatias/tratamento farmacológico , Proteínas tau/metabolismoRESUMO
Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region of the heavy chain (CDR H3) is the region of highest sequence diversity and is formed by the joining of heavy chain VH, DH and JH germline segments combined with random nucleotide trimming and additions between these segments. We show that CDR H3 and junctional segment length distributions are biased in human antibody repertoires as a function of VH, VL and JH germline segment utilization. Most length biases are apparent in the naive and antigen experienced B cell compartments but not in nonproductive recombination products, indicating B cell selection as a major driver of these biases. Our findings reveal biases in the antibody CDR H3 diversity landscape shaped by VH, VL, and JH germline segment use during naive and antigen-experienced repertoire selection.
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Diversidade de Anticorpos/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , HumanosRESUMO
Antibodies from B-cell clonal lineages share sequence and structural properties as well as epitope specificity. Clonally unrelated antibodies can similarly share sequence and specificity properties and are said to be convergent. Convergent antibody responses against several antigens have been described in humans and mice and include different classes of shared sequence features. In particular, some antigens and epitopes can induce convergent responses of clonally unrelated antibodies with restricted heavy (VH) and light (VL) chain variable region germline segment usage without similarity in the heavy chain third complementarity-determining region (CDR H3), a critical specificity determinant. Whether these V germline segment-restricted responses reflect a general epitope specificity restriction of antibodies with shared VH/VL pairing is not known. Here, we investigated this question by determining patterns of antigen binding competition between clonally unrelated antigen-specific rat antibodies from paired-chain deep sequencing datasets selected based solely on VH/VL pairing. We found that antibodies with shared VH/VL germline segment pairings but divergent CDR H3 sequences almost invariably have restricted epitope specificity indicated by shared binding competition patterns. This epitope restriction included 82 of 85 clonally unrelated antibodies with 13 different VH/VL pairings binding in 8 epitope groups in 2 antigens. The corollary that antibodies with shared VH/VL pairing and epitope-restricted binding can accommodate widely divergent CDR H3 sequences was confirmed by in vitro selection of variants of anti-human epidermal growth factor receptor 2 antibodies known to mediate critical antigen interactions through CDR H3. Our results show that restricted epitope specificity determined by VH/VL germline segment pairing is a general property of rodent antigen-specific antibodies.