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HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including Type1 Diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble α and ß chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of inter-chain disulfide engineering by introducing paired cysteines to juxtaposed positions in the α and ß chains of HLA-DQ7, encoded by HLA-DQA1*05:01 and HLA-DQB1*03:01 respectively. We identified three variant peptide-HLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83C-E5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, high-affinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents.
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Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of â¼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.
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Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Animais , Humanos , Bacteriófago M13 , Técnicas de Visualização da Superfície Celular , Células HEK293 , Ligação ProteicaRESUMO
OBJECTIVES: Haemophilia B (HB), characterised by deficient factor IX (FIX), leads to spontaneous bleeds. Severe cases require prophylactic FIX replacement. This post hoc analysis assessed the first spontaneous bleeds among previously untreated patients (PUPs) with HB treated with recombinant FIX Fc fusion protein (rFIXFc) (NCT02234310) to identify factors influencing bleeds. METHODS: Subjects included paediatric PUPs with HB (≤2 IU/dL endogenous FIX). Analyses described treatment patterns (on demand [OD] vs. prophylaxis) and prophylaxis type (started on vs. switched to prophylaxis). Kaplan-Meier analyses assessed the time to first spontaneous bleed, including median time to event and fitting models with predictors for treatment regimen and/or baseline age. RESULTS: PUPs B-LONG enrolled 33 subjects. Baseline age did not influence the time to first spontaneous bleed for any rFIXFc regimen. Those who started on prophylaxis with rFIXFc (n = 11), compared with those treated OD (n = 22), had an extended time to first spontaneous bleed. Starting prophylaxis afforded a 93% reduced risk of first spontaneous bleed versus starting OD (hazard ratio [95% confidence interval]: 0.071 [0.009-0.592]) (p = .015). CONCLUSION: rFIXFc prophylaxis, particularly starting early, reduced the risk of bleeding and delayed time to first spontaneous bleed compared with rFIXFc OD. Hence, initial treatment regimens impact bleed patterns in paediatric PUPs.
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OBJECTIVE: Manufacturing changes occur commonly throughout stages of biologics development and may result in product quality attribute changes. As changes in critical quality attributes have the potential to affect clinical safety and efficacy of products, it is imperative to ensure the quality and clinical performance before introducing the after-change products. Thus, we embarked on this project to understand what data have supported the manufacturing changes for licensed products with pre- and post-approval changes. METHODS: We surveyed the manufacturing changes of 85 monoclonal antibodies and 10 Fc fusion proteins approved by the Food and Drug Administration as of December 25, 2021. After collecting the type and timing of changes for these products, we investigated the approaches that provided supporting data for the changes. The source documents included reports submitted by applicants and FDA's regulatory reviews. RESULTS: Analytical comparability was assessed to support all identified manufacturing changes. Supporting clinical data were available in 92% of these manufacturing changes; including data from pharmacokinetic comparability studies alone (3%), other studies on efficacy or safety (70%) and a combination of both (19%). Clinical pharmacokinetic comparability data contributed to supporting substantial changes, such as host cell type or master cell bank changes, concentration or formulation changes, and changes from pre-filled syringes to autoinjectors, especially when introduced after completing pivotal studies. CONCLUSION: Our comprehensive retrospective analysis provides an understanding of the regulatory experience and industry practice, which could facilitate developing appropriate comparability approaches to support manufacturing changes in the future.
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Anticorpos Monoclonais , Estados Unidos , Estudos Retrospectivos , Previsões , United States Food and Drug AdministrationRESUMO
The growing complexity of novel biopharmaceutical formats, such as Fc-fusion proteins, in increasingly competitive environment has highlighted the need of high-throughput analytical platforms. Multi-attribute method (MAM) is an emerging analytical technology that utilizes liquid chromatography coupled with mass spectrometry to monitor critical quality attributes (CQAs) in biopharmaceuticals. MAM is intended to supplement or replace the conventional chromatographic and electrophoretic approaches used for quality control and drug release purpose. In this investigation, we have developed an agile sample preparation approach for deploying MAM workflow for a complex VEGFR-targeted therapeutic Fc-fusion protein. Initially, a systematic time course evaluation of tryptic digestion step was performed to achieve maximum amino acid sequence coverage of >96.5%, in a short duration of 2 h, with minimum assay artifacts. This approach facilitated precise identification of five sites of N-glycosylation with successful monitoring of other CQAs such as deamidation, oxidation, etc. Subsequently, the developed MAM workflow with suitable tryptic digestion time was qualified according to the International council for harmonisation (i.e. ICH) Q2R1 guidelines for method validation. Post-validation, the analytical workflow was also evaluated for its capability to identify unknown moieties, termed as 'New Peak Detection' (i.e. NPD), and assess fold change between the reference and non-reference samples, in a representative investigation of pH stress study. The study, thus, demonstrated the suitability of the MAM workflow for characterization of heavily glycosylated Fc-fusion proteins. Moreover, its NPD feature could offer an all-encompassing view if applied for forced degradation and stability studies.
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Produtos Biológicos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Glicosilação , Fluxo de TrabalhoRESUMO
Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.
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Glicoproteínas , Cloreto de Sódio , Cricetinae , Animais , Glicosilação , Cricetulus , Glicoproteínas/metabolismo , Polissacarídeos/química , Células CHORESUMO
There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.
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Produtos Biológicos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Dissulfetos/químicaRESUMO
OBJECTIVE: To evaluate the lifetime cost-effectiveness of recombinant factor IX Fc fusion protein (rFIXFc) and recombinant factor IX (rFIX) for the treatment of hemophilia B (HB) in China. METHODS: We developed a decision-analytic Markov model including three health states: alive, requiring surgery, and dead. This model estimated the lifetime cost and quality-adjusted life-years (QALYs) of prophylaxis in childhood, followed by on-demand treatment in adulthood for moderate-severe to severe HB patients from China's healthcare system perspective. Efficacy data derived from pivotal clinical trials, clinical guideline recommendations, and expert consultation were applied to two scenarios (full dose and low dose). One-way sensitivity analysis and probabilistic sensitivity analysis (PSA) were performed to assess the robustness of the model. OUTCOMES: Lifetime cost, QALYs, and the incremental cost-effectiveness ratio were calculated, and the results were compared with willingness-to-pay (WTP) thresholds of one to three times the gross domestic product per capita of China in 2021 ($12,551-$37,653). RESULTS: RFIXFc was associated with lower cost and more QALYs than rFIX in both scenarios, which suggested that it is a dominant strategy (more effective and cheaper) for moderate-severe to severe HB in China. In the full-dose scenario, rFIXFc saved more money and yielded more QALYs than in the low-dose scenario (low doses are the typical clinical reality in China). PSA demonstrated that rFIXFc had an over 90% probability of being cost-effective with full-dose and low-dose treatment at WTP thresholds of $12,551-$37,653. CONCLUSIONS: Compared with rFIX, rFIXFc appears to be a cost-effective option for the lifetime management of moderate-severe to severe HB patients in China.
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Hemofilia A , Hemofilia B , Humanos , Fator IX/metabolismo , Fator IX/uso terapêutico , Hemofilia B/tratamento farmacológico , Análise de Custo-Efetividade , Hemofilia A/tratamento farmacológico , China , Análise Custo-BenefícioRESUMO
KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.
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Vacinas Anticâncer , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Vacinas Anticâncer/uso terapêutico , Imunidade , Próstata/metabolismo , Antígeno Prostático Específico , Neoplasias da Próstata/terapiaRESUMO
The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography-tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography-tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography-mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography-tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan® , etc., can display pivotal analytical capabilities in automated liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.
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Medicamentos Biossimilares , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Espectrometria de Massas/métodos , Anticorpos Monoclonais/química , Glicosilação , Polissacarídeos/químicaRESUMO
BACKGROUND: IgA nephropathy is a common primary glomerulonephritis caused by mesangial deposition of poly-IgA complexes. The disease follows a variable course of clinical progression, with a high risk of kidney failure. Although no specific therapy is available, enzymatic strategies to clear IgA deposits are being considered for the treatment of rapidly progressive IgA nephropathy. METHODS: We chose an IgA protease of commensal bacterium Clostridium ramosum, termed AK183, as the template for constructing a recombinant biologic. To extend the t1/2 in blood, we fused AK183 to the Fc segment of human IgG1. Activities of this Fc-AK183 fusion protein toward the cleavage and subsequent clearance of IgA were tested in mouse models. RESULTS: First, we discovered an autocleavage activity of AK183 that separates the N-terminal protease from its C-terminal autotransporter ß domain. Therefore, we grafted Fc to the N terminus of AK183 and demonstrated its week-long enzymatic activity in mice. In addition, the proteolytic fragments of IgA generated in the reaction with Fc-AK183 were effectively removed from circulation via kidney filtration. The combined actions of Fc-AK183-mediated cleavage and subsequent renal clearance of IgA resulted in a lasting obliteration of blood IgA, as demonstrated in a human IgA-injection model and in a humanized α1KI transgenic model. Fc-AK183 was also able to remove chronic IgA and associated complement C3 deposits in the glomerulus. CONCLUSION: We constructed a chimeric fusion of IgA protease with Fc and demonstrated its long-lasting efficacy as a promising targeted therapy for IgA nephropathy in mouse models.
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Glomerulonefrite por IGA , Animais , Modelos Animais de Doenças , Feminino , Firmicutes , Glomerulonefrite por IGA/terapia , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G , Masculino , Camundongos , Receptores Fc , Serina EndopeptidasesRESUMO
FcγR binding characterization is one of the critical attributes during the development of therapeutic antibodies. Here, we report a novel assay format to characterize IgG-FcγR interaction in the presence of antigen using Surface plasmon resonance (SPR). The new assay format was developed by creating stable antigen/antibody immunocomplexes on a sensor chip surface before injection of FcγRs. In this assay format, binding activity of both huIgG1 (including IgG1 Fc fusion Protein) and huIgG2 increased significantly to most activating human FcγRs, especially to FcγRI, FcγRIIa-131H and FcγRIIIa-158F. To our knowledge, this study provides the first set of evidence using a biophysical method to demonstrate antigen binding facilitating IgG-FcγR interaction, especially for huIgG2 where previous studies did not indicate its binding to human FcγRI or FcγRIIIa-158F. Although further studies are needed to investigate the correlation of the binding data with effector function data in vivo, our results suggest that it may be useful to evaluate the IgG-FcγR interaction in the presence of antigen to help design safer and more effective biotherapeutics.
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Receptores de IgGRESUMO
INTRODUCTION: Perioperative dosing recommendations vary across Nordic haemophilia treatment centres (HTCs) for extended half-life (EHL) factor concentrates in haemophilia A/B (HA/HB) patients. AIM: To summarise Nordic real-world surgical experiences with EHL recombinant factor VIII/IX Fc (rFVIIIFc/rFIXFc) fusion proteins using retrospective data from clinical records at four HTCs in Finland, Sweden and Norway. METHODS: Factor dosing and surgical outcomes were recorded from HA/HB patients who underwent surgery and were treated with rFVIIIFc/rFIXFc. Perioperative factor dosing regimens were clinician-determined based on local practises. RESULTS: Twenty five surgeries were performed on 20 patients, all covered by bolus injections except one minor HA surgery; eight minor surgeries were in paediatric patients. Median preoperative rFVIIIFc dose for major HA surgeries (n = 8) was 48 IU/kg (range: 35-57), with total consumption up to Day 14 of 427 IU/kg (196-568). For the two major HB surgeries (in one patient), preoperative rFIXFc doses were 50 IU/kg and 20 IU/kg; total consumption up to Day 14 was 130 IU/kg and 40 IU/kg. Median preoperative rFVIIIFc/rFIXFc bolus doses for minor HA (n = 10) and HB (n = 4) surgeries were 50 IU/kg (24-79) and 47 IU/kg (40-71), with total consumption up to Day 5 of 138 IU/kg (49-404) and 100 IU/kg (43-125), respectively. Intraoperative and postoperative haemostatic responses were rated as at least good/excellent for 24/25 surgeries, with bleeding episodes reported in only three surgeries. CONCLUSION: Nordic real-world experiences suggest that EHL products can be used safely and effectively for peri-operative haemostasis. Further research is required to develop local dosing guidelines for optimised treatment schedules.
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Hemofilia A , Hemofilia B , Criança , Fator IX/uso terapêutico , Fator VIII/uso terapêutico , Meia-Vida , Hemofilia A/tratamento farmacológico , Hemofilia A/cirurgia , Hemofilia B/tratamento farmacológico , Hemofilia B/cirurgia , Hemorragia/tratamento farmacológico , Humanos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Estudos Retrospectivos , Países Escandinavos e Nórdicos , Resultado do TratamentoRESUMO
Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.
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Biossíntese de Proteínas , RNA , Cricetinae , Animais , Cricetulus , Células CHO , RNA/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: ⢠An in vitro screening system for synthetic signal peptide in mammalian cells was established ⢠This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay ⢠The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.
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Peptídeos , Sinais Direcionadores de Proteínas , Animais , Células CHO , Cricetinae , Cricetulus , Peptídeos/química , Peptídeos/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
CTLA4-haploinsufficiency is a complex disease of immune dysregulation presenting with a broad spectrum of clinical manifestations. CTLA4-Fc fusion proteins such as abatacept have been described to alleviate immune dysregulation in several adult cases of CTLA4-haploinsufficiency. However, until now only few cases of pediatric CTLA4-haploinsufficiency treated with abatacept have been described. Here we present two pediatric cases of severe CTLA4-haploinsufficiency refractory to conventional immunosuppressive therapies that responded rapidly to treatment with abatacept. No side effects were observed during a follow-up period of 7-15 months. While one patient has successfully undergone HSCT the second patient continues to receive abatacept. Our cases demonstrate safe medium-term use of abatacept in the pediatric population.
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Abatacepte/uso terapêutico , Antígeno CTLA-4/deficiência , Imunossupressores/uso terapêutico , Adolescente , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Feminino , Haploinsuficiência/genética , Haploinsuficiência/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/terapia , Masculino , Mutação de Sentido Incorreto , Linfócitos T Reguladores/imunologiaRESUMO
Prophylaxis with factor VIII (FVIII) is the current therapeutic approach for people with haemophilia A. However, standard half-life (SHL) FVIII products must be injected frequently, imposing a substantial burden on the individual and making it difficult to tailor therapy according to patient need and lifestyle, which could impact adherence. Recombinant FVIII Fc fusion protein (rFVIIIFc; Elocta® , Sobi; Eloctate® , Sanofi) is a recombinant fusion protein that undergoes slower clearance from the body than SHL FVIII products. This pharmacokinetic property of rFVIIIFc allows prophylactic administration every 3-5 days, or once weekly in selected patients, with doses adjusted to patient needs and clinical outcomes. Higher FVIII levels can be achieved maintaining dosing frequency similar to that usually applied with SHL FVIII. This review provides a summary of recent data from the A-LONG, Kids A-LONG, ASPIRE and PUPs A-LONG studies and recently published real-world experience relevant to rFVIIIFc use in individualised regimens. The review also introduces ongoing studies of rFVIIIFc, including its use for induction of immune tolerance, and discusses some aspects to consider when switching patients to rFVIIIFc and managing ongoing treatment. In summary, rFVIIIFc is suitable for individualised prophylaxis regimens that can be tailored according to patient clinical needs and lifestyle.
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Fator VIII , Hemofilia A , Tolerância Imunológica/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas , Proteínas Recombinantes de Fusão , Ensaios Clínicos como Assunto , Fator VIII/farmacocinética , Fator VIII/uso terapêutico , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3'-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. KEY POINTS: ⢠EBNA-1-amplified HEK293 cells were established using gene amplification system. ⢠EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. ⢠EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein.
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Infecções por Vírus Epstein-Barr , Amplificação de Genes , Animais , Células CHO , Cricetinae , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Metotrexato , Plasmídeos/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
FcRn plays a major role in regulating immune homeostasis, but it is also able to transport biologics across cellular barriers. The question of whether FcRn could be an efficient transporter of biologics across the nasal epithelial barrier is of particular interest, as it would allow a less invasive strategy for the administration of biologics in comparison to subcutaneous, intramuscular or intravenous administrations, which are often used in clinical practice. A focused systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. It was registered on the international prospective register of systematic reviews PROSPERO, which helped in identifying articles that met the inclusion criteria. Clinical and preclinical studies involving FcRn and the nasal delivery of biologics were screened, and the risk of bias was assessed across studies using the Oral Health Assessment Tool (OHAT). Among the 12 studies finally included in this systematic review (out of the 758 studies screened), 11 demonstrated efficient transcytosis of biologics through the nasal epithelium. Only three studies evaluated the potential toxicity of biologics' intranasal delivery, and they all showed that it was safe. This systematic review confirmed that FcRn is expressed in the nasal airway and the olfactory epithelium, and that FcRn may play a role in IgG and/or IgG-derived molecule-transcytosis across the airway epithelium. However, additional research is needed to better characterize the pharmacokinetic and pharmacodynamic properties of biologics after their intranasal delivery.
Assuntos
Produtos Biológicos/administração & dosagem , Antígenos de Histocompatibilidade Classe I/metabolismo , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Receptores Fc/metabolismo , Animais , Produtos Biológicos/metabolismo , Transporte Biológico , Biomarcadores , Sistemas de Liberação de Medicamentos , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ligação Proteica , Receptores Fc/química , Receptores Fc/genética , TranscitoseRESUMO
As an essential modulator of IgG disposition, the neonatal Fc receptor (FcRn) governs the pharmacokinetics and functions many therapeutic modalities. In this review, we thoroughly reexamine the hitherto elucidated biological and thermodynamic properties of FcRn to provide context for our assessment of more recent advances, which covers antigen-binding fragment (Fab) determinants of FcRn affinity, transgenic preclinical models, and FcRn targeting as an immune-complex (IC)-clearing strategy. We further comment on therapeutic antibodies authorized for treating SARS-CoV-2 (bamlanivimab, casirivimab, and imdevimab) and evaluate their potential to saturate FcRn-mediated recycling. Finally, we discuss modeling and simulation studies that probe the quantitative relationship between in vivo IgG persistence and in vitro FcRn binding, emphasizing the importance of endosomal transit parameters.