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Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Camundongos , Animais , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Fatores de Crescimento Endotelial/uso terapêutico , Acuidade Visual , Transtornos da Visão/complicações , Transtornos da Visão/tratamento farmacológico , Cegueira/complicaçõesRESUMO
The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above â¼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.
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Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFß2 and TGFß3 (Sun et al. in Sci Transl Med, 2021. https://doi.org/10.1126/scitranslmed.abe0407 ). Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFß2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFß2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFß2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 (Kabat numbering) of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.
Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais Humanizados/química , Fragmentos Fab das Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Fator de Crescimento Transformador beta2/química , Fator de Crescimento Transformador beta3/química , Animais , Anticorpos Monoclonais Humanizados/genética , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Estrutura Quaternária de Proteína , Coelhos , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/genéticaRESUMO
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.
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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
Fusion of biologic therapeutics to hyaluronic acid binding proteins, such as the link domain (LD) of Tumor necrosis factor (TNF)-Stimulated Gene-6 (TSG-6), is expected to increase vitreous residence time following intravitreal injection and provide for long-acting delivery. The toxicity of a single intravitreal dose of free TSG-6-LD and fusion proteins of TSG-6-LD and a nonbinding rabbit antibody fragment (RabFab) were assessed in New Zealand White rabbits. Animals administered free TSG-6-LD exhibited extensive lens opacities and variable retinal vascular attenuation, correlated with microscopic findings of lens and retinal degeneration. Similar but less severe findings were present in animals dosed with the RabFab-TSG-6-LD fusion proteins. In-life ocular inflammation was noted in all animals from 7-days postdose and was associated with high anti-RabFab antibody titers in animals administered fusion proteins. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, and hypertrophy and migration of vimentin, glial fibrillary acidic protein, and glutamine synthetase positive Müller cells to the outer nuclear layer. Further assessment of alternative hyaluronic acid binding protein fusions should consider the potential for retinal degeneration and enhanced immune responses early in development.
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Retina , Degeneração Retiniana , Animais , Proteína Glial Fibrilar Ácida , Injeções Intravítreas , Coelhos , Degeneração Retiniana/induzido quimicamenteRESUMO
PURPOSE: Several neonatal Fc receptor (FcRn) variants of an anti-tumor necrosis factor (TNF)-α humanized monoclonal IgG antibodies (mAbs) were developed but the effect of their differential FcRn binding affinities on pharmacokinetic (PK) behavior were difficult to be definitively measured in vivo due to formation of anti-therapeutic antibody (ATA). A semi-mechanistic model was developed to investigate the quantitative relationship between the FcRn binding affinity and PK of mAbs in cynomolgus monkey with the presence of ATA. METHODS: PK and ATA data from cynomolgus monkeys which received a single intravenous dose of adalimumab, wild-type or two FcRn variant (N434H and N434A) anti-TNF-α mAbs were included in the analysis. Likelihood-based censored data handling method was used to include many PK observations with BQL values for model development. A fully integrated PK-ATA model was developed and used to fit simultaneously to the PK/ATA data. RESULTS AND CONCLUSIONS: The PK and ATA time-profiles and effect of FcRn-binding affinity on PK of mAbs were well described by the model and the parameters were estimated with good precision. The model was used successfully to construct quantitative relationships between FcRn binding affinity and PK of anti-TNF-α mAbs in the presence of the ATA-mediated elimination and interferences.
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Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Antígenos de Histocompatibilidade Classe I/imunologia , Ligação Proteica/imunologia , Receptores Fc/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Macaca fascicularisRESUMO
The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-alpha monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentration-time curve from time 0 to day 7 than the WT (749 +/- 71.9 and 819 +/- 81.5 versus 592 +/- 56.8 microg/ml . day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.
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Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/farmacocinética , Receptores Fc/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Disponibilidade Biológica , Humanos , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Injeções Subcutâneas , Macaca fascicularis , Camundongos , Camundongos SCID , Especificidade da EspécieRESUMO
CAPS (Ca(2+)-dependent activator protein for secretion) functions in priming Ca(2+)-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca(2+)-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca(2+) in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.
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Proteínas de Ligação ao Cálcio/fisiologia , Caseína Quinase II/química , Exocitose , Sequência de Aminoácidos , Animais , Células COS , Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Humanos , Camundongos , Dados de Sequência Molecular , Células PC12 , Fosforilação , Ratos , Homologia de Sequência de Aminoácidos , Serina/química , Triazóis/farmacologiaRESUMO
A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2-4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 framework-specific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNF(NC)). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNF(NC) Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities.
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Anticorpos Anti-Idiotípicos/sangue , Anticorpos Monoclonais/farmacocinética , Técnicas Eletroquímicas/métodos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais Humanizados , Técnicas Eletroquímicas/instrumentação , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Células Jurkat , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Macaca fascicularis , Masculino , Testes de Neutralização , Sensibilidade e EspecificidadeRESUMO
Five CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-A crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor.
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Receptores Imunológicos/química , Receptores do Fator de Necrose Tumoral/química , Receptores Virais/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Luz , Linfócitos/imunologia , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Complexos Multiproteicos/química , Mutagênese Insercional , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Membro 14 de Receptores do Fator de Necrose Tumoral , Receptores Virais/genética , Receptores Virais/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/químicaRESUMO
Immune cell cosignaling receptors are important modulators of immune cell function. For T cells, cosignaling receptors supply necessary secondary signals supporting activation or attenuation after engagement of antigen-presenting cells. The primary cosignaling receptors belong to either the Ig (CD28-like) or TNF receptor (TNFR) superfamilies. The CD28 family is comprised of coinhibitory and costimulatory receptors. The three coinhibitory receptors are cytotoxic T lymphocyte antigen 4, programmed death-1, and B and T lymphocyte attenuator (BTLA). Although cytotoxic T lymphocyte antigen 4 and programmed death-1 interact with B7-Ig family counter receptors, the ligand for BTLA is less clear. From a protein-protein interaction screen, we identified the TNFR family member herpesvirus entry mediator (HVEM) as a counter receptor for BTLA. Here we show that HVEM binds BTLA with high affinity and can form a ternary complex with its known ligands homologous to lymphotoxin, showing inducible expression, and competing with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT) or lymphotoxin alpha and BTLA. In addition, binding of HVEM to BTLA attenuates T cell activation, identifying HVEM/BTLA as a coinhibitory receptor pair. This study is a demonstration of a direct interaction between the primary T cell cosignaling receptors of the CD28 and TNFR families.
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Receptores Imunológicos/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Animais , Humanos , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores Imunológicos/química , Receptores do Fator de Necrose Tumoral/química , Membro 14 de Receptores do Fator de Necrose Tumoral , Receptores Virais/química , Linfócitos T/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Ca2+-dependent activator protein for secretion (CAPS) is a cytosolic protein essential for the Ca2+-dependent fusion of dense-core vesicles (DCVs) with the plasma membrane and the regulated secretion of a subset of neurotransmitters. The mechanism by which CAPS functions in exocytosis and the means by which it associates with target membranes are unknown. We identified two domains in CAPS with distinct membrane-binding properties that were each essential for CAPS activity in regulated exocytosis. The first of these, a centrally located pleckstrin homology domain, exhibited three properties: charge-based binding to acidic phospholipids, binding to plasma membrane but not DCV membrane, and stereoselective binding to phosphatidylinositol 4,5-bisphosphate. Mutagenesis studies revealed that the former two properties but not the latter were essential for CAPS function. The central pleckstrin homology domain may mediate transient CAPS interactions with the plasma membrane during Ca2+-triggered exocytosis. The second membrane association domain comprising distal C-terminal sequences mediated CAPS targeting to and association with neuroendocrine DCVs. The CAPS C-terminal domain was also essential for optimal activity in regulated exocytosis. The presence of two membrane association domains with distinct binding specificities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion.