RESUMEN

Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50 values of the designed antibody mutants are decreased by up to 17 fold, and KD values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.

Asunto(s)

Afinidad de Anticuerpos , Redes Neurales de la Computación , Humanos , Anticuerpos/inmunología , Anticuerpos/química , Simulación por Computador , Aprendizaje Profundo , Antígenos/inmunología , Unión Proteica , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares

RESUMEN

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Asunto(s)

Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Humanos , Transglutaminasas/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Inmunoprecipitación/métodos , Anticuerpos/inmunología , Proteínas de Unión al GTP/inmunología

RESUMEN

Droplet microfluidics has become a very powerful tool in high-throughput screening, including antibody discovery. Screens are usually carried out by physically sorting droplets hosting cells of the desired phenotype, breaking them, recovering the encapsulated cells, and sequencing the paired antibody light and heavy chain genes at the single-cell level. This series of multiple consecutive manipulation steps of rare screening hits is complex and challenging, resulting in a significant loss of clones with the desired phenotype or large fractions of cells with incomplete antibody information. Here, we present fluorescence-activated droplet sequencing, in which droplets showing the desired phenotype are selectively picoinjected with reagents for RT-PCR. Subsequently, light and heavy chain genes are natively paired, fused into a single-chain fragment variant format, and amplified before off-chip transfer and downstream nanopore sequencing. This workflow is sufficiently sensitive for obtaining different paired full-length antibody sequences from as little as five droplets, fulfilling the desired phenotype. Replacing physical sorting by specific sequencing overcomes a general bottleneck in droplet microfluidic screening and should be compatible with many more applications.

Asunto(s)

Anticuerpos , Humanos , Microfluídica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos

RESUMEN

Leukemia inhibitory factor (LIF) is involved in the progression of different cancers. In this study, we investigated the effect of anti-LIF antibodies on immune-related gene expression in the Balb/c mouse model of breast cancer. To immunize mice against LIF, recombinant LIF with Freund adjuvant was injected into the test group, whereas the control group received phosphate-buffered saline with adjuvant. Tumor induction (4T1 cell line) was performed by increasing the antibody titer. The expression of immune-related genes was evaluated by real-time PCR. The anti-LIF titer was significantly increased in the immunized group. The expression of genes related to the differentiation of T helper (Th)-1, Th-2, and Th-17 cells was significantly higher in the immunized group than in the control group. In addition, anti-LIF did not have a significant effect on the expression of genes related to the differentiation of regulatory T cells, and immune checkpoint-associated genes. Additionally, the test group had higher survival and lower tumor development rates. The results demonstrated that the anti-LIF antibody may potentially play a role in the differentiation of immune cells or immune responses. However, further studies utilizing advanced techniques are necessary to validate its function.

Asunto(s)

Neoplasias de la Mama , Factor Inhibidor de Leucemia , Ratones Endogámicos BALB C , Animales , Femenino , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/metabolismo , Factor Inhibidor de Leucemia/inmunología , Ratones , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Anticuerpos/inmunología

RESUMEN

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Asunto(s)

Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Proteína Huntingtina , Inmunoprecipitación , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/inmunología , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunología , Animales , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/genética , Células HEK293

RESUMEN

Neuromuscular blocking agents (NMBAs) relax skeletal muscles to facilitate surgeries and ease intubation but can lead to adverse reactions, including complications because of postoperative residual neuromuscular blockade (rNMB) and, in rare cases, anaphylaxis. Both adverse reactions vary between types of NMBAs, with rocuronium, a widely used nondepolarizing NMBA, inducing one of the longest rNMB durations and highest anaphylaxis incidences. rNMB induced by rocuronium can be reversed by the synthetic γ-cyclodextrin sugammadex. However, in rare cases, sugammadex can provoke anaphylaxis. Thus, additional therapeutic options are needed. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies. To understand the pathogenesis of rocuronium-induced anaphylaxis and to identify potential therapeutics, we investigated the memory B cell antibody repertoire of patients with suspected hypersensitivity to rocuronium. We identified polyclonal antibody repertoires with a high diversity among V(D)J genes without evidence of clonal groups. When recombinantly expressed, these antibodies demonstrated specificity and low affinity for rocuronium without cross-reactivity for other NMBAs. Moreover, when these antibodies were expressed as human immunoglobulin E (IgE), they triggered human mast cell activation and passive systemic anaphylaxis in transgenic mice, although their affinities were insufficient to serve as reversal agents. Rocuronium-specific, high-affinity antibodies were thus isolated from rocuronium-immunized mice. The highest-affinity antibody was able to reverse rocuronium-induced neuromuscular blockade in nonhuman primates with kinetics comparable to that of sugammadex. Together, these data support the hypothesis that antibodies cause anaphylactic reactions to rocuronium and pave the way for improved diagnostics and neuromuscular blockade reversal agents.

Asunto(s)

Anafilaxia , Rocuronio , Rocuronio/efectos adversos , Animales , Humanos , Anafilaxia/inmunología , Anticuerpos , Ratones , Periodo Perioperatorio , Androstanoles/efectos adversos , Sugammadex/efectos adversos , Inmunoglobulina E/inmunología , Especificidad de Anticuerpos , Femenino , Modelos Animales de Enfermedad , Masculino

RESUMEN

One of the important genes for eyespot development in butterfly wings is Distal-less. Its function has been evaluated via several methods, including CRISPR/Cas9 genome editing. However, functional inhibition may be performed at the right time at the right place using a different method. Here, we used a novel protein delivery method for pupal wing tissues in vivo to inactivate a target protein, Distal-less, with a polyclonal anti-Distal-less antibody using the blue pansy butterfly Junonia orithya. We first demonstrated that various antibodies including the anti-Distal-less antibody were delivered to wing epithelial cells in vivo in this species. Treatment with the anti-Distal-less antibody reduced eyespot size, confirming the positive role of Distal-less in eyespot development. The treatment eliminated or deformed a parafocal element, suggesting a positive role of Distal-less in the development of the parafocal element. This result also suggested the integrity of an eyespot and its corresponding parafocal element as the border symmetry system. Taken together, these findings demonstrate that the antibody-mediated protein knockdown method is a useful tool for functional assays of proteins, such as Distal-less, expressed in pupal wing tissues, and that Distal-less functions for eyespots and parafocal elements in butterfly wing color pattern development.

Asunto(s)

Anticuerpos , Mariposas Diurnas , Proteínas de Insectos , Alas de Animales , Animales , Mariposas Diurnas/metabolismo , Mariposas Diurnas/genética , Alas de Animales/metabolismo , Alas de Animales/crecimiento & desarrollo , Anticuerpos/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Pigmentación/genética , Técnicas de Silenciamiento del Gen

RESUMEN

Small molecules find extensive application in medicine, food safety, and environmental studies, particularly in biomedicine. Immunoassay technology, leveraging the specific recognition between antigens and antibodies, offers a superior alternative to traditional physical and chemical analysis methods. This approach allows for the rapid and accurate detection of small molecular compounds, owing to its high sensitivity, specificity, and swift analytical capabilities. However, small molecular compounds often struggle to effectively stimulate an immune response due to their low molecular weight, weak antigenicity, and limited antigenic epitopes. To overcome this, coupling small molecule compounds with macromolecular carriers to form complete antigens is typically required to induce specific antibodies in animals. Consequently, the preparation of small-molecule artificial antigens and the production of efficient specific antibodies are crucial for achieving precise immunoassays. This paper reviews recent advancements in small molecule antibody preparation technology, emphasizing the design and synthesis of haptens, the coupling of haptens with carriers, the purification and identification of artificial antigens, and the preparation of specific antibodies. Additionally, it evaluates the current technological shortcomings and limitations while projecting future trends in artificial antigen synthesis and antibody preparation technology.

Asunto(s)

Anticuerpos , Antígenos , Haptenos , Antígenos/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/química , Haptenos/química , Haptenos/inmunología , Humanos , Inmunoensayo/métodos

RESUMEN

Traditional computational methods for antibody design involved random mutagenesis followed by energy function assessment for candidate selection. Recently, diffusion models have garnered considerable attention as cutting-edge generative models, lauded for their remarkable performance. However, these methods often focus solely on the backbone or sequence, resulting in the incomplete depiction of the overall structure and necessitating additional techniques to predict the missing component. This study presents Antibody-SGM, an innovative joint structure-sequence diffusion model that addresses the limitations of existing protein backbone generation models. Unlike previous models, Antibody-SGM successfully integrates sequence-specific attributes and functional properties into the generation process. Our methodology generates full-atom native-like antibody heavy chains by refining the generation to create valid pairs of sequences and structures, starting with random sequences and structural properties. The versatility of our method is demonstrated through various applications, including the design of full-atom antibodies, antigen-specific CDR design, antibody heavy chains optimization, validation with Alphafold3, and the identification of crucial antibody sequences and structural features. Antibody-SGM also optimizes protein function through active inpainting learning, allowing simultaneous sequence and structure optimization. These improvements demonstrate the promise of our strategy for protein engineering and significantly increase the power of protein design models.

Asunto(s)

Cadenas Pesadas de Inmunoglobulina , Modelos Moleculares , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Ingeniería de Proteínas , Regiones Determinantes de Complementariedad/química , Conformación Proteica , Anticuerpos/química , Anticuerpos/inmunología

RESUMEN

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

Asunto(s)

Estreptavidina , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos/metabolismo , Humanos , Biotinilación , Microscopía Fluorescente/métodos , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo

RESUMEN

The comprehensive understanding of the orientation of antibodies on a solid surface is crucial for affinity-based sensing mechanisms. In this study, we demonstrated that the orientation of primary antibodies modified on carboxy-functionalized polystyrene (PS) particles can be analyzed using zeta potential behavior at different pH based on the combined Gouy-Chapman-Stern model and the acid dissociation of carboxy groups and antibodies. We observed that at low surface concentrations of the primary antibody, a side-on orientation was predominant. However, at higher concentrations (approximately 30000 antibodies per PS particle), the orientation shifted to an end-on type due to steric hindrance. Furthermore, the reaction mechanism of the secondary antibody exhibited pH-dependent behavior. At pH > 7, the zeta potential changes were attributed to the antibody-antibody reaction, whereas at pH < 7, adsorption of secondary antibody onto the PS particle was observed, leading to a change in the orientation of the primary antibody modified on the PS particle to an end-on type. The change in zeta potential due to secondary antibody binding indicated a detection limit of 37000 antibodies per PS particle. As a result, we revealed that the analysis of zeta potential behavior enables the evaluation of antibody orientation and the detection of zeptomole order antibodies. This study represents the first demonstration of this capability. We anticipate that the present concept and results will broaden the quantitative application of zeta potential measurements and have significant implications for research areas, including physical chemistry and analytical chemistry.

Asunto(s)

Anticuerpos , Poliestirenos , Poliestirenos/química , Concentración de Iones de Hidrógeno , Anticuerpos/química , Anticuerpos/inmunología , Propiedades de Superficie , Tamaño de la Partícula

RESUMEN

Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle (n = 8), or the anti-MBG antibody (n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC50 = 0.28 µmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

Asunto(s)

Bufanólidos , Fibrosis , Ratas Sprague-Dawley , Insuficiencia Renal Crónica , Vasodilatación , Animales , Masculino , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Vasodilatación/efectos de los fármacos , Ratas , Bufanólidos/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Anticuerpos/farmacología , Nefrectomía , Nitroprusiato/farmacología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Colágeno Tipo I/metabolismo , Endotelina-1/metabolismo

RESUMEN

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 µM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

Asunto(s)

Técnicas Biosensibles , Exosomas , MicroARNs , Humanos , Exosomas/química , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , MicroARNs/aislamiento & purificación , MicroARNs/genética , Células MCF-7 , Anticuerpos/inmunología , Anticuerpos/química

RESUMEN

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Asunto(s)

Anticuerpos , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Sinaptotagmina I , Sinaptotagmina I/inmunología , Sinaptotagmina I/metabolismo , Humanos , Citometría de Flujo/métodos , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunología

RESUMEN

Macroporous three-dimensional (3D) framework structured melamine foam-based Enzyme-Linked Immunosorbent Assay (f-ELISA) biosensors were developed for rapid, reliable, sensitive, and on-site detection of trace amount of biomolecules and chemicals. Various ligands can be chemically immobilized onto the melamine foam, which brings in the possibility of working with antibodies, nanobodies, and peptides, respectively, as affinity probes for f-ELISA biosensors with improved stability. Different chemical reagents can be used to modify the foam materials, resulting in varied reactivities with antibodies, nanobodies, and peptides. As a result, the f-ELISA sensors produced from these modified foams exhibit varying levels of sensitivity and performance. This study demonstrated that the chemical reagents used for immobilizing antibodies, nanobodies, and peptides could affect the sensitivities of the f-ELISA sensors, and their storage stabilities under different temperatures varied depending on the sensing probes used, with f-ELISA sensors employing nanobodies as probes exhibiting the highest stability. This study not only showcases the versatility of the f-ELISA system but also opens new avenues for developing cost-effective, portable, and user-friendly diagnostic tools with optimized sensitivity and stability.

Asunto(s)

Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Anticuerpos de Dominio Único , Triazinas , Triazinas/análisis , Triazinas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Técnicas Biosensibles/métodos , Péptidos/química , Anticuerpos/inmunología , Anticuerpos/química , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/química , Límite de Detección

RESUMEN

This study aimed to investigate whether class A1 scavenger receptor (SR-A1) regulated macrophage polarization and gut microbial alteration during intestinal inflammation of colitis. A murine colitis model was established by feeding with dextran sulfate sodium (DSS), and treatment groups were injected intravenously with SR-A1 antibody. Results showed a preventive effect on colitis symptoms and fewer inflammatory cell infiltrates in treatment groups. Down-regulation of inflammatory cytokines and up-regulation of anti-inflammatory cytokine related to macrophages were seen in murine PBMC and LPMC after injected with SR-A1 antibody. The percentage of M2 macrophages was also elevated in treatment groups. In addition, SR-A1 antibody treatment resulted in the decreased apoptosis and increased proliferation of colonic epithelial cells. Other findings indicated that SR-A1 antibody injection could mediate its anti-inflammatory effect via inhibiting TLR4-MyD88-NF-kB signaling pathway and alterating the gut microbiota composition. Our research identified SR-A1 as a potential therapeutic target in inflammatory bowel disease (IBD).

Asunto(s)

Colitis , Microbioma Gastrointestinal , Macrófagos , Receptores Depuradores de Clase A , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Colitis/inmunología , Colitis/inducido químicamente , Colitis/microbiología , Colitis/metabolismo , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores Depuradores de Clase A/metabolismo , Sulfato de Dextran/toxicidad , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Modelos Animales de Enfermedad , Citocinas/metabolismo , Anticuerpos , FN-kappa B/metabolismo , Ratones Endogámicos C57BL , Masculino , Apoptosis/efectos de los fármacos

RESUMEN

Monotherapy, especially the use of antibodies targeting vascular endothelial growth factor (VEGF), has shown limitations in treating choroidal neovascularization (CNV) since reactive oxygen species (ROS) also exacerbate CNV formation. Herein, we developed a combination therapy based on a DNA origami platform targeting multiple components of ocular neovascularization. Our study demonstrated that ocular neovascularization was markedly suppressed by intravitreal injection of a rectangular DNA origami sheet modified with VEGF aptamers (Ap) conjugated to an anti-VEGF antibody (aV) via matrix metalloproteinase (MMP)-cleavable peptide linkers in a mouse model of CNV. Typically, the DNA origami-based therapeutic platform selectively accumulates in neovascularization lesions owing to the dual-targeting ability of the aV and Ap, followed by the cleavage of the peptide linker by MMPs to release the antibody. Together, the released antibody and Ap inhibited VEGF activity. Moreover, the residual bare DNA origami could effectively scavenge ROS, reducing oxidative stress at CNV sites and thus maximizing the synergistic effects of inhibiting neovascularization.

Asunto(s)

Neovascularización Coroidal , ADN , Factor A de Crecimiento Endotelial Vascular , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Animales , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/química , ADN/química , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/química , Anticuerpos/química

RESUMEN

The effective treatment of acute lung injury (ALI) remains a significant challenge. Patients with ALI demonstrate an abundance of proinflammatory mediators in both bronchoalveolar lavage fluid (BALF) and circulating plasma. Bardoxolone methyl (BM) is a semi-synthetic triterpenoid derived from oleanolic acid, a natural product known for its ability to inhibit proinflammatory signaling. GSDMD is a signaling protein involved in pyroptosis, a form of programmed cell death. It has been reported that its upstream proteins play a role in the pathogenesis of ALI. However, there is currently no research examining whether the effect of BM on the occurrence and development of ALI is associated with changes in GSDMD protein. In this study, we prepared nanostructured lipid carriers loaded with BM and conjugated with anti-PECAM-1 antibody (PECAM@BM NLCs). PECAM@BM NLCs were designed to specifically bind to pulmonary vascular endothelial cells that highly express the PECAM-1 receptors. We also aimed to investigate the protective effects of PECAM@BM NLCs on ALI and elucidate the underlying molecular mechanisms. The results demonstrated that PECAM@BM NLCs accumulated in the lung tissues and significantly alleviated the inflammatory injury of ALI. This was evidenced by the changes in the lung wet/dry ratio, the total protein concentration, proinflammatory cytokines in BALF, and the histopathological progress. Additionally, we elucidated that PECAM@BM NLCs had the ability to inhibit the assembly of NLRP3 inflammasome and pro-caspase-1 complex, thereby suppressing the induction of pyroptosis. This mechanism resulted in the inhibition of N-terminal GSDMD expression and effectively prevented the progression of ALI.

Asunto(s)

Lesión Pulmonar Aguda , Pulmón , Nanoestructuras , Ácido Oleanólico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Ácido Oleanólico/farmacología , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/química , Animales , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/patología , Nanoestructuras/química , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Portadores de Fármacos/química , Masculino , Ratones , Neumonía/tratamiento farmacológico , Neumonía/patología , Neumonía/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratones Endogámicos C57BL , Lípidos/química , Anticuerpos/farmacología , Líquido del Lavado Bronquioalveolar/química , Humanos , Sistemas de Liberación de Medicamentos/métodos , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos

RESUMEN

The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus.

Asunto(s)

Administración Intranasal , Antidepresivos , Antígenos CD , Factor Neurotrófico Derivado del Encéfalo , Depresión , Hipocampo , Proteína del Gen 3 de Activación de Linfocitos , Sistema de Señalización de MAP Quinasas , Estrés Psicológico , Animales , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Masculino , Antidepresivos/farmacología , Antidepresivos/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Depresión/tratamiento farmacológico , Antígenos CD/metabolismo , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Anticuerpos/farmacología , Carbazoles/farmacología , Carbazoles/administración & dosificación , Transducción de Señal/efectos de los fármacos , Alcaloides Indólicos

RESUMEN

The ability to accurately predict antibody-antigen complex structures from their sequences could greatly advance our understanding of the immune system and would aid in the development of novel antibody therapeutics. There have been considerable recent advancements in predicting protein-protein interactions (PPIs) fueled by progress in machine learning (ML). To understand the current state of the field, we compare six representative methods for predicting antibody-antigen complexes from sequence, including two deep learning approaches trained to predict PPIs in general (AlphaFold-Multimer and RoseTTAFold), two composite methods that initially predict antibody and antigen structures separately and dock them (using antibody-mode ClusPro), local refinement in Rosetta (SnugDock) of globally docked poses from ClusPro, and a pipeline combining homology modeling with rigid-body docking informed by ML-based epitope and paratope prediction (AbAdapt). We find that AlphaFold-Multimer outperformed other methods, although the absolute performance leaves considerable room for improvement. AlphaFold-Multimer models of lower quality display significant structural biases at the level of tertiary motifs (TERMs) toward having fewer structural matches in non-antibody-containing structures from the Protein Data Bank (PDB). Specifically, better models exhibit more common PDB-like TERMs at the antibody-antigen interface than worse ones. Importantly, the clear relationship between performance and the commonness of interfacial TERMs suggests that the scarcity of interfacial geometry data in the structural database may currently limit the application of ML to the prediction of antibody-antigen interactions.

Asunto(s)

Complejo Antígeno-Anticuerpo , Complejo Antígeno-Anticuerpo/química , Conformación Proteica , Anticuerpos/química , Anticuerpos/inmunología , Simulación del Acoplamiento Molecular , Modelos Moleculares , Humanos