Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.479
Filtrar
1.
J Chromatogr A ; 1689: 463730, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36592480

RESUMO

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association. For this reason, a Self-Association SMA (SAS-SMA) model was applied, which was extended by two activity coefficients for the salt and protein in solution. This model is able to describe protein-protein interactions in the form of self-dimerization and thus can describe anti-Langmuir elution behavior. Linear gradient elution (LGE) experiments were carried out to obtain a broad dataset ranging from pH 4.5 to 7.3 and from 50 to 375 mmol/L Na+ for model parameter determination. High loading LGE experiments were conducted with an increasing load from 0.5 up to 75.0 mgbsAb/mLresin. Thereby, pH-dependent empirical correlations for the activity coefficient of the solute protein, for the equilibrium constant of the self-dimerization process and for the shielding factor could be set up and ultimately incorporated into the SAS-SMA model. This pH-dependent SAS-SMA model was thus able to simulate anti-Langmuir behavior over extended ranges of pH, counterion concentration, and column loading. The model was confirmed by experimental verification of simulated linear pH gradient elutions up to a load of 75.0 mgbsAb/mLresin.


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Cromatografia por Troca Iônica , Anticorpos Monoclonais/metabolismo , Cloreto de Sódio , Cátions , Resinas de Troca de Cátion , Concentração de Íons de Hidrogênio
2.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675043

RESUMO

Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and other bioactive cargos. We also highlighted various applications and advantages plant-produced vaccines have and their relevance for treating human and animal illnesses. Furthermore, we summarized the plant-based biologics that have passed through clinical trials, the unique challenges they faced, and the challenges they will face to qualify, become available, and succeed on the market.


Assuntos
Agricultura Molecular , Vírus de Plantas , Animais , Humanos , Plantas Geneticamente Modificadas/metabolismo , Vacinas Sintéticas , Vírus de Plantas/genética , Anticorpos Monoclonais/metabolismo
3.
Sci Immunol ; 8(79): eadd4947, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36638191

RESUMO

The PD-1 receptor triggers a negative immunoregulatory mechanism that prevents overactivation of immune cells and subsequent inflammatory diseases. Because of its biological significance, PD-1 has been a drug target for modulating immune responses. Immunoenhancing anti-PD-1 blocking antibodies have become a widely used cancer treatment; however, little is known about the required characteristics for anti-PD-1 antibodies to be capable of stimulating immunosuppressive activity. Here, we show that PD-1 agonists exist in the group of anti-PD-1 antibodies recognizing the membrane-proximal extracellular region in sharp contrast to the binding of the membrane-distal region by blocking antibodies. This trend was consistent in an analysis of 81 anti-human PD-1 monoclonal antibodies. Because PD-1 agonist antibodies trigger immunosuppressive signaling by cross-linking PD-1 molecules, Fc engineering to enhance FcγRIIB binding of PD-1 agonist antibodies notably improved human T cell inhibition. A PD-1 agonist antibody suppressed inflammation in murine disease models, indicating its clinical potential for treatment of various inflammatory disorders, including autoimmune diseases.


Assuntos
Anticorpos Monoclonais , Linfócitos T , Animais , Camundongos , Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Humanos
4.
Sci Transl Med ; 15(678): eabo0205, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36630481

RESUMO

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.


Assuntos
Anemia Aplástica , Doença Enxerto-Hospedeiro , Camundongos , Animais , Linfócitos T/metabolismo , Anemia Aplástica/metabolismo , Citocinas/metabolismo , Transdução de Sinais , Anticorpos Monoclonais/metabolismo , Doença Enxerto-Hospedeiro/metabolismo
5.
Sci Rep ; 13(1): 808, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36646875

RESUMO

Glioblastoma is characterized by a strong self-renewal potential and poor differentiated state. We have reported previously that the (pro)renin receptor [(P)RR] is a potential target for glioma therapy by silencing the (P)RR gene. Here, we have examined the effects of a monoclonal antibody against (P)RR on gliomagenesis. Human glioma cell lines (U251MG and U87MG) and a glioma stem cell line (MGG23) were used for the in vitro study. The expressions of the Wnt/ß-catenin signaling pathway (Wnt signaling pathway) components and stemness markers were measured by Western blotting. The effects of the (P)RR antibody on cell proliferation, sphere formation, apoptosis and migration were also examined. Subcutaneous xenografts were also examined in nude mice. Treatment with the (P)RR antibody reduced expression of Wnt signaling pathway components and stemness markers. Furthermore, the (P)RR antibody reduced cell proliferation and decreased sphere formation significantly. The treatment also suppressed migration and induced apoptosis. In a subcutaneous xenograft model, systemic administration of the (P)RR antibody reduced tumor volume significantly. These data show that treatment with the (P)RR antibody is a potential therapeutic strategy for treating glioblastoma.


Assuntos
Glioblastoma , Glioma , Camundongos , Animais , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Anticorpos Monoclonais/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Glioma/genética , Via de Sinalização Wnt/genética , Proliferação de Células , beta Catenina/metabolismo , Regulação Neoplásica da Expressão Gênica
6.
J Biomed Sci ; 29(1): 108, 2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36550570

RESUMO

BACKGROUND: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harbor diverse spike (S) protein sequences, which can greatly influence the efficacies of therapeutics. Therefore, it would be of great value to develop neutralizing monoclonal antibodies (mAbs) that can broadly recognize multiple variants. METHODS: Using an mRNA-LNP immunization strategy, we generated several mAbs that specifically target the conserved S2 subunit of SARS-CoV-2 (B-S2-mAbs). These mAbs were assessed for their neutralizing activity with pseudotyped viruses and binding ability for SARS-CoV-2 variants. RESULTS: Among these mAbs, five exhibited strong neutralizing ability toward the Gamma variant and also recognized viral S proteins from the Wuhan, Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.2 and BA.5) variants. Furthermore, we demonstrated the broad reactivities of these B-S2-mAbs in several different applications, including immunosorbent, immunofluorescence and immunoblotting assays. In particular, B-S2-mAb-2 exhibited potent neutralization of Gamma variant (IC50 = 0.048 µg/ml) in a pseudovirus neutralization assay. The neutralizing epitope of B-S2-mAb-2 was identified by phage display as amino acid residues 1146-1152 (DSFKEEL) in the S2 subunit HR2 domain of SARS-CoV-2. CONCLUSION: Since there are not many mAbs that can bind the S2 subunit of SARS-CoV-2 variants, our set of B-S2-mAbs may provide important materials for basic research and potential clinical applications. Importantly, our study results demonstrate that the viral S2 subunit can be targeted for the production of cross-reactive antibodies, which may be used for coronavirus detection and neutralization.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Anticorpos Antivirais , Anticorpos Monoclonais/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes
7.
Molecules ; 27(24)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36557993

RESUMO

Therapeutic proteins have unique advantages over small-molecule drugs in the treatment of various diseases, such as higher target specificity, stronger pharmacological efficacy and relatively low side effects. These advantages make them increasingly valued in drug development and clinical practice. However, although highly valued, the intrinsic limitations in their physical, chemical and pharmacological properties often restrict their wider applications. As one of the most important post-translational modifications, glycosylation has been shown to exert positive effects on many properties of proteins, including molecular stability, and pharmacodynamic and pharmacokinetic characteristics. Glycoengineering, which involves changing the glycosylation patterns of proteins, is therefore expected to be an effective means of overcoming the problems of therapeutic proteins. In this review, we summarize recent efforts and advances in the glycoengineering of erythropoietin and IgG monoclonal antibodies, with the goals of illustrating the importance of this strategy in improving the performance of therapeutic proteins and providing a brief overview of how glycoengineering is applied to protein-based drugs.


Assuntos
Anticorpos Monoclonais , Engenharia de Proteínas , Glicosilação , Anticorpos Monoclonais/metabolismo , Processamento de Proteína Pós-Traducional , Imunoglobulina G/química , Polissacarídeos/metabolismo
8.
Protein Eng Des Sel ; 352022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36336952

RESUMO

The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.


Assuntos
Sistemas CRISPR-Cas , Humanos , /metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Edição de Genes , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo
9.
Microvasc Res ; 139: 104228, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547346

RESUMO

The subcutaneous injection has emerged to become a feasible self-administration practice for biotherapeutics due to the patient comfort and cost-effectiveness. However, the available knowledge about transport and absorption of these agents after subcutaneous injection is limited. Here, a mathematical framework to study the subcutaneous drug delivery of mAbs from injection to lymphatic uptake is presented. A three-dimensional poroelastic model is exploited to find the biomechanical response of the tissue by taking into account tissue deformation during the injection. The results show that including tissue deformability noticeably changes tissue poromechanical response due to the significant dependence of interstitial pressure on the tissue deformation. Moreover, the importance of the amount of lymph fluid at the injection site and the injection rate on the drug uptake to lymphatic capillaries is highlighted. Finally, variability of lymphatic uptake due to uncertainty in parameters including tissue poromechanical and lymphatic absorption parameters is evaluated. It is found that interstitial pressure due to injection is the major contributing factor in short-term lymphatic absorption, while the amount of lymph fluid at the site of injection determines the long-term absorption of the drug. Finally, it is shown that the lymphatic uptake results are consistent with experimental data available in the literature.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Linfa/metabolismo , Modelos Biológicos , Absorção Fisiológica , Anticorpos Monoclonais/metabolismo , Transporte Biológico , Simulação por Computador , Difusão , Módulo de Elasticidade , Humanos , Injeções Subcutâneas , Análise Numérica Assistida por Computador , Porosidade , Pressão , Autoadministração , Fatores de Tempo
10.
Sci Rep ; 12(1): 19562, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36380063

RESUMO

Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.


Assuntos
Anticorpos Monoclonais , Canais de Cátion TRPM , Camundongos , Animais , Humanos , Epitopos , Anticorpos Monoclonais/metabolismo , Sequência de Aminoácidos , Peptídeos/metabolismo , Mutação de Sentido Incorreto , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
11.
FASEB J ; 36(12): e22629, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36349990

RESUMO

ß1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active ß1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of ß1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting ß1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.


Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Animais , Humanos , Camundongos , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Integrina beta1/metabolismo , Anticorpos Monoclonais/metabolismo , Integrinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo
12.
Cells ; 11(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36359801

RESUMO

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.


Assuntos
Antígenos Ly , Neutrófilos , Camundongos , Animais , Neutrófilos/metabolismo , Antígenos Ly/metabolismo , Camundongos SCID , Anticorpos Monoclonais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos BALB C
13.
Protein Sci ; 31(12): e4486, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36317676

RESUMO

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.


Assuntos
Antígeno B7-H1 , Bacteriófagos , Animais , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biblioteca de Peptídeos , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Anticorpos Monoclonais/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Especificidade de Anticorpos
14.
Cell Rep ; 41(4): 111555, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36288706

RESUMO

Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.


Assuntos
Interleucina-17 , Receptores de Interleucina-17 , Humanos , Camundongos , Animais , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Fibronectinas/metabolismo , Ligantes , Anticorpos Neutralizantes/metabolismo , Regulação Neoplásica da Expressão Gênica , Carcinogênese , Anticorpos Monoclonais/metabolismo
15.
J Am Chem Soc ; 144(42): 19485-19498, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36222719

RESUMO

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.


Assuntos
Anticorpos Monoclonais , Imunoadsorventes , Humanos , Camundongos , Animais , LDL-Colesterol , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/metabolismo , Peptídeos/farmacologia , Colesterol , Albuminas , Albumina Sérica Humana , Subtilisinas , Pró-Proteína Convertase 9
16.
Sci Rep ; 12(1): 17047, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36220879

RESUMO

COVID-19 is the global pandemic that affected our population in the past 2 years. Considerable research has been done to better understand the pathophysiology of this disease and to identify new therapeutic targets, especially for severe cases. Galectin-3 (Gal-3) is a receptor present at the surface of different cell types, namely epithelial and inflammatory cells, which has been described as a severity marker in COVID-19. The activation of Gal-3 through its binding protein (Gal-3BP) is directly linked to the production of pro-inflammatory cytokines that contribute for the cytokine storm (CS) observed in severe COVID-19 patients. Here, we show that D2, a recombinant fragment of the lectin-binding region of Gal-3BP was able to stimulate the expression of IL-6 in colon and lung epithelial cell lines in ß-galactoside dependent manner. We further show that D2-induced IL-6 augmentation was reduced by the anti-Gal-3BP monoclonal antibody 1959. Our data confirm and extend prior findings of Gal-3BP mediated IL-6 induction, enlightening the potential of its antibody-mediated s blockage for the prevention and treatment of CS and severe disease in COVID-19 patients.


Assuntos
SARS-CoV-2 , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Proteínas de Transporte , Linhagem Celular , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Galectina 3/metabolismo , Humanos , Interleucina-6/metabolismo
17.
Front Immunol ; 13: 1003986, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36203585

RESUMO

Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a+ and CD163+ and B cells based on the expression of CD79a. NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αß T cells were classified by their CD8ß+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8ß++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine.


Assuntos
Pesquisa Biomédica , Fator de Necrose Tumoral alfa , Animais , Anticorpos Monoclonais/metabolismo , Células Matadoras Naturais , Modelos Animais , Perforina/metabolismo , Suínos , Porco Miniatura , Fator de Necrose Tumoral alfa/metabolismo
18.
J Cancer Res Ther ; 18(5): 1409-1416, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36204890

RESUMO

Background: Tumor-associated macrophages (TAMs) are vital to the tumor microenvironment. They are classified as antitumor M1-type or protumor M2-type macrophages. M2-type macrophages accumulate in the tumor stroma and are related to poor prognosis. Iron oxide nanoparticles are used as drug delivery vehicles because of the structure of carboxyl groups on their surface and their ability to be easily phagocytosed by macrophages. Aim: The signal transducer and activator of transcription 6 (STAT6) signaling pathway controls M2 macrophage polarization, but the STAT6 signaling pathway inhibitor AS1517499 lacks efficient targeting in vivo. Thus, our study aimed to block the polarization of TAMs to M2-type macrophages. Methods and Material: We used ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as drug carriers coated with the STAT6 signaling pathway inhibitors AS1517499 and CD163 monoclonal antibodies to synthesize the targeted nanocomplex AS1517499-USPION-CD163 utilizing the carbodiimide method. Then, we determined its physicochemical properties, including hydrodynamic size distribution, ultrastructure, iron concentration, protein content and activity of the CD163 monoclonal antibody, AS1517499 content, and selectivity for M2-type macrophages, and its biological applications. Results: The hydrodynamic size distribution was stable (average size = 95.37 nm). Regarding biological applications, the targeted nanocomplex selectively inhibited M2-type macrophages. Conclusions: The targeted nanocomplex AS1517499-USPION-CD163 showed high selectivity for M2-type macrophages. Therefore, iron oxide nanoparticles targeting TAMs may be an effective approach to TAM therapy.


Assuntos
Antineoplásicos , Microambiente Tumoral , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbodi-Imidas/metabolismo , Carbodi-Imidas/farmacologia , Portadores de Fármacos , Compostos Férricos , Humanos , Ferro/metabolismo , Macrófagos/metabolismo , Fator de Transcrição STAT6/metabolismo , Fator de Transcrição STAT6/farmacologia
19.
Int J Mol Sci ; 23(20)2022 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-36293075

RESUMO

Systemic lupus erythematosus (SLE) is characterized by the breakdown of self-tolerance, the production of high-affinity pathogenic autoantibodies and derailed B cell responses, which indicates the importance of central players, such as follicular T helper (TFH) subsets and follicular T regulatory (TFR) cells, in the pathomechanism of the disease. In this study, we aimed to analyze the distribution of the circulating counterparts of these cells and their association with disease characteristics and B cell disproportions in SLE. We found that the increased percentage of activated circulating TFH (cTFH) and cTFR cells was more pronounced in cutaneous lupus; however, among cTFH subsets, the frequency of cTFH17 cells was decreased in patients with lupus nephritis. Furthermore, the decreased proportion of cTFH17 cells was associated with low complement C4 levels and high disease activity scores. We also investigated whether the blocking of the IL-21 receptor (IL-21R) with an anti-IL-21R monoclonal antibody inhibits the B cell response, since IL-21 primarily produced by TFH cells potentially promotes humoral immunity. We observed that anti-IL-21R inhibited plasmablast generation and immunoglobulin production. Our study demonstrated that, besides cTFR/cTFH imbalance, cTFH17 cells play a crucial role in SLE pathogenesis, and modulating cTFH-B cell interaction through the IL-21/IL-21R pathway may be a promising therapeutic strategy to suppress the pathological B cell response.


Assuntos
Lúpus Eritematoso Sistêmico , Receptores de Interleucina-21 , Humanos , Receptores de Interleucina-21/metabolismo , Linfócitos T Auxiliares-Indutores , Linfócitos T Reguladores , Autoanticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Complemento C4/metabolismo
20.
Front Immunol ; 13: 989895, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36300122

RESUMO

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.


Assuntos
Mieloma Múltiplo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Camundongos , Animais , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Cetuximab/metabolismo , Linfócitos T CD8-Positivos/patologia , Camundongos SCID , Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras Naturais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Mieloma Múltiplo/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...