RESUMO
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologiaRESUMO
Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
Assuntos
Anticorpos , Especificidade de Anticorpos , Proteínas de Membrana , Proteólise , Ubiquitina-Proteína Ligases , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias Colorretais/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.
Assuntos
Neoplasias da Mama , Linfócitos T CD8-Positivos , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Animais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Feminino , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Receptor ErbB-2/imunologia , Linhagem Celular Tumoral , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismoRESUMO
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.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Sinucleinopatias , Camundongos , Animais , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Doenças Neurodegenerativas/metabolismo , Sinucleinopatias/patologia , Neurônios Dopaminérgicos/metabolismoRESUMO
PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.
Assuntos
Neoplasias , Anticorpos de Domínio Único , Humanos , Linfócitos T CD8-Positivos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias/diagnóstico por imagem , Linhagem Celular TumoralRESUMO
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.
Assuntos
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
BACKGROUND: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding. RESULTS: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals. CONCLUSIONS: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.
Assuntos
Asma , Degeneração Macular , Bioensaio , Biomarcadores , Desenvolvimento de Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Interleucina-33 , Sensibilidade e EspecificidadeRESUMO
Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.
Assuntos
Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Morte Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Feminino , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , UbiquitinaçãoRESUMO
The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.
Assuntos
Antibacterianos/farmacologia , Anticorpos Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
BACKGROUND: Atopic dermatitis (AD) and psoriasis are common inflammatory diseases canonically described as involving distinct T(H) polarization and granulocytic infiltration. Acute AD lesions are associated with T(H)2 and eosinophilic inflammation, whereas psoriatic lesions are associated with T(H)1/T(H)17 and neutrophilic inflammation. Despite intensive investigation, these pathways remain incompletely understood in vivo in human subjects. OBJECTIVE: Using AD and psoriatic lesional skin as exemplar T(H)2 and T(H)1/T(H)17 diseased tissue, we sought to clarify common and unique molecular and pathophysiologic features in inflamed skin with different types of inflammatory polarization. METHODS: We conducted gene expression microarray analyses to identify distinct and commonly dysregulated expression in AD (based on Hanifin and Rajka criteria) and psoriatic lesions. We defined gene sets (GSs) as comprising genes encoding cytokines, chemokines, and growth factors that were uniquely or jointly dysregulated in patients with AD and those with psoriasis and calculated aggregate GS expression scores for lesional skin of patients with these dermatoses and healthy control skin. RESULTS: The atopic dermatitis gene set (AD-GS) score correlated with systemic and local measures of allergic inflammation, including serum IgE levels, blood eosinophil counts, and tissue eosinophil counts. Unexpectedly, genes encoding neutrophil chemoattractants among the common GS were highly expressed in AD lesional skin. Hematoxylin and eosin and immunohistochemical analyses showed the numbers of neutrophils in AD lesional skin were comparable with those in psoriatic lesional skin, and both were correlated with the extent of expression of neutrophil chemoattractant genes. CONCLUSION: These data are evidence that neutrophilic inflammation is a feature of lesional AD pathology comorbid with allergic inflammation.
Assuntos
Quimiocinas/metabolismo , Dermatite Atópica/imunologia , Neutrófilos/imunologia , Psoríase/imunologia , Pele/imunologia , Adulto , Idoso , Movimento Celular , Quimiocinas/genética , Dermatite Atópica/genética , Feminino , Humanos , Imunoglobulina E/sangue , Mediadores da Inflamação/metabolismo , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Psoríase/genética , Equilíbrio Th1-Th2 , Transcriptoma , Adulto JovemRESUMO
Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c(-/-)) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c(-)(/)(-) splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c(+/+)) cells. In contrast, T cell development and function were largely normal in Sh2d3c(-/-) mice. The thymi of Sh2d3c(-/-) mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c(-/-) mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos T-Independentes/imunologia , Subpopulações de Linfócitos B/patologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Feminino , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transdução de Sinais/genética , Baço/patologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O2â¢-), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.
Assuntos
NADPH Oxidases , Superóxidos , Humanos , Fragmentos de Imunoglobulinas , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/química , Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Estudo de Prova de Conceito , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/metabolismo , Células RAW 264.7 , RatosRESUMO
Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.
Assuntos
Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3 , Animais , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Camundongos , Isoformas de Proteínas/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Antineoplásicos/imunologia , Imunoconjugados/imunologia , Oligopeptídeos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Feminino , Proteínas Ligadas por GPI/imunologia , Células HEK293 , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Camundongos SCID , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologiaRESUMO
Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.
Assuntos
Anticorpos Monoclonais/farmacologia , Citocinas/imunologia , Hipersensibilidade Imediata/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Ligante OX40/antagonistas & inibidores , Células Th2/imunologia , Inibidores do Fator de Necrose Tumoral , Animais , Anticorpos Monoclonais/uso terapêutico , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Humanos , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/patologia , Imunoglobulina E/imunologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Pulmão/imunologia , Pulmão/patologia , Macaca mulatta , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ligante OX40/imunologia , Receptores OX40/imunologia , Pele/imunologia , Pele/patologia , Células Th2/patologia , Fatores de Necrose Tumoral/imunologia , Linfopoietina do Estroma do TimoRESUMO
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.
Assuntos
Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Sequência de Aminoácidos , Animais , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , RatosRESUMO
Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.
Assuntos
Anticorpos Monoclonais/metabolismo , Linfócitos B/imunologia , Região Variável de Imunoglobulina/genética , Doença de Parkinson/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Células Clonais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/imunologia , Hipermutação Somática de ImunoglobulinaRESUMO
Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.