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Status dystonicus is the most severe form of dystonia with life-threatening complications if not treated promptly. We present consensus recommendations for the initial management of acutely worsening dystonia (including pre-status dystonicus and status dystonicus), as well as refractory status dystonicus in children. This guideline provides a stepwise approach to assessment, triage, interdisciplinary treatment, and monitoring of status dystonicus. The clinical pathways aim to: (1) facilitate timely recognition/triage of worsening dystonia, (2) standardize supportive and dystonia-directed therapies, (3) provide structure for interdisciplinary cooperation, (4) integrate advances in genomics and neuromodulation, (5) enable multicenter quality improvement and research, and (6) improve outcomes. © 2024 International Parkinson and Movement Disorder Society.
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BACKGROUND: Adaptor protein complex 4-associated hereditary spastic paraplegia (AP-4-HSP) is caused by pathogenic biallelic variants in AP4B1, AP4M1, AP4E1, and AP4S1. OBJECTIVE: The aim was to explore blood markers of neuroaxonal damage in AP-4-HSP. METHODS: Plasma neurofilament light chain (pNfL) and glial fibrillary acidic protein (GFAP) levels were measured in samples from patients and age- and sex-matched controls (NfL: n = 46 vs. n = 46; GFAP: n = 14 vs. n = 21) using single-molecule array assays. Patients' phenotypes were systematically assessed using the AP-4-HSP natural history study questionnaires, the Spastic Paraplegia Rating Scale, and the SPATAX disability score. RESULTS: pNfL levels increased in AP-4-HSP patients, allowing differentiation from controls (Mann-Whitney U test: P = 3.0e-10; area under the curve = 0.87 with a 95% confidence interval of 0.80-0.94). Phenotypic cluster analyses revealed a subgroup of individuals with severe generalized-onset seizures and developmental stagnation, who showed differentially higher pNfL levels (Mann-Whitney U test between two identified clusters: P = 2.5e-6). Plasma GFAP levels were unchanged in patients with AP-4-HSP. CONCLUSIONS: pNfL is a potential disease marker in AP-4-HSP and can help differentiate between phenotypic subgroups. © 2023 International Parkinson and Movement Disorder Society.
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Complexo 4 de Proteínas Adaptadoras , Paraplegia Espástica Hereditária , Humanos , Complexo 4 de Proteínas Adaptadoras/genética , Paraplegia Espástica Hereditária/genética , Filamentos Intermediários/metabolismo , Fenótipo , MutaçãoRESUMO
Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
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Produtos Biológicos/química , Quimiocina CXCL13/antagonistas & inibidores , Modelos Moleculares , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos , Produtos Biológicos/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Humanos , Cinética , Mutação , Agregados Proteicos , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Solubilidade , Difração de Raios XRESUMO
Wormian bones are independent ossification centers found within cranial sutures or fontanelles. Though common in adult populations, their presence in children can be associated with several conditions such as osteogenesis imperfecta, hypothyroidism, pyknodysostosis, cleidocranial dysostosis, rickets, and acrocallosal syndrome. These conditions encompass a large range of clinical features but there has only been 1 other reported patient of exomphalos occurring concurrently with these ossicles. The authors present the case of a child with an anterior fontanellar Wormian bone, dysmorphic facial features, and exomphalos major born to unaffected parents. The pattern of features seen in this child did not closely match any condition commonly associated with Wormian bones. The only other reported case of both Wormian bone and exomphalos was in a child with acrocallosal syndrome who presented with more severe dysmorphic features than seen here. It is possible that this patient represents a previously unknown association between acrocallosal syndrome and exomphalos or a less severe variant of the condition. Conversely, this patient may possibly illustrate a newly discovered association between Wormian bones, facial dysmorphism, and midline abdominal defects.
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Anormalidades Múltiplas/diagnóstico , Fontanelas Cranianas/anormalidades , Suturas Cranianas/anormalidades , Anormalidades Craniofaciais/diagnóstico , Hérnia Umbilical/diagnóstico , Atrofia Muscular/diagnóstico , Anormalidades Múltiplas/cirurgia , Fontanelas Cranianas/diagnóstico por imagem , Suturas Cranianas/diagnóstico por imagem , Anormalidades Craniofaciais/cirurgia , Humanos , Recém-Nascido , Masculino , Atrofia Muscular/cirurgiaRESUMO
BACKGROUND: The Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees. RESULTS: After confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect. CONCLUSIONS: Our data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.
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Abelhas/genética , Comportamento Animal/fisiologia , Resistência à Doença/genética , Sistemas Neurossecretores , Agricultura , Animais , Abelhas/parasitologia , Genótipo , Humanos , Larva , Nosema/patogenicidade , Odorantes , Polinização/genética , Transdução de Sinais/genética , Varroidae/genética , Varroidae/patogenicidadeRESUMO
Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C(H)3-C(H)3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.
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Fragmentos Fc das Imunoglobulinas , Engenharia de Proteínas , Animais , Linhagem Celular , Glicosilação , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Receptores Fc/genética , Receptores Fc/metabolismoRESUMO
A distinct subset of acute myeloid leukemia (AML) is characterized by the presence of the Philadelphia chromosome (Ph+), due to reciprocal translocation t(9;22)(q34;q11.2). This chromosomal rearrangement leads to the fusion of the breakpoint cluster region (BCR) gene on chromosome 22 with the ABL1 gene on chromosome 9, generating the BCR::ABL1 fusion gene. The Ph+ AML subtype is associated with poor prognosis and resistance to conventional chemotherapy. Beyond the well-established BCR::ABL1 fusion, recent studies have shed light on additional genetic abnormalities in Ph+ AML, including associations with rearrangements involving core binding factor beta (CBFB). We describe a case of de novo AML with concurrent BCR::ABL1 and CBFB::MYH11 rearrangements.
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BACKGROUND AND AIMS: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations to the CF transmembrane conductance regulator (CFTR). Symptoms and severity of the disease can be quite variable suggesting modifier genes play an important role. MATERIALS AND METHODS: Exome sequencing was performed on six individuals carrying homozygous deltaF508 for CFTR genotype but present with rapidly progressing CF (RPCF). Data was analyzed using an unbiased genome-wide genetic burden test against 3076 controls. Single cell RNA sequencing data from LungMAP was utilized to evaluate unique and co-expression of candidate genes, and structural modeling to evaluate the deleterious effects of identified candidate variants. RESULTS: We have identified solute carrier family 26 member 9 (SLC26A9) as a modifier gene to be associated with RPCF. Two rare missense SLC26A9 variants were discovered in three of six individuals deemed to have RPCF: c.229G > A; p.G77S (present in two patients), and c.1885C > T; p.P629S. Co-expression of SLC26A9 and CFTR mRNA is limited across different lung cell types, with the highest level of co-expression seen in human (6.3 %) and mouse (9.0 %) alveolar type 2 (AT2) cells. Structural modeling suggests deleterious effects of these mutations as they are in critical protein domains which might affect the anion transport capability of SLC26A9. CONCLUSION: The enrichment of rare and potentially deleterious SLC26A9 mutations in patients with RPCF suggests SLC26A9 may act as an alternative anion transporter in CF and is a modifier gene associated with this lung phenotype.
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Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Homozigoto , Mutação , Transportadores de Sulfato , Humanos , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismo , Feminino , Masculino , Antiporters/genética , Antiporters/química , Animais , CamundongosRESUMO
Children with developmental and epileptic encephalopathies often present with co-occurring dyskinesias. Pathogenic variants in ARX cause a pleomorphic syndrome that includes infantile epilepsy with a variety of movement disorders ranging from focal hand dystonia to generalized dystonia with frequent status dystonicus. In this report, we present three patients with severe movement disorders as part of ARX-associated epilepsy-dyskinesia syndrome, including a patient with a novel pathogenic missense variant (p.R371G). These cases illustrate diagnostic and management challenges of ARX-related disorder and shed light on broader challenges concerning epilepsy-dyskinesia syndromes.
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Proteínas de Homeodomínio , Transtornos dos Movimentos , Fatores de Transcrição , Humanos , Masculino , Feminino , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/diagnóstico , Transtornos dos Movimentos/etiologia , Pré-Escolar , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Lactente , Mutação de Sentido Incorreto , CriançaRESUMO
Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293FTM and ExpiCHO-STM with the transfection reagents ExpiFectamineTM and polyethylenimine. We discovered that there are significant differences between Expi293FTM and ExpiCHO-STM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.
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Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation. Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.
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This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.
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Imunoglobulina G , Células Matadoras Naturais , Humanos , Animais , Camundongos , Imunoglobulina G/genética , MamíferosRESUMO
Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.
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Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Esporos Bacterianos/fisiologia , Alanina/química , Alanina/metabolismo , Aminoácidos/química , Bacillus subtilis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , MutaçãoRESUMO
Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody-like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multi-chain, bi-specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In-depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi-chain molecule.
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Cricetulus , Animais , Células CHO , Cricetinae , Proteínas Recombinantes/genética , Estudos Retrospectivos , Transfecção , TransgenesRESUMO
GDF15 is a distant TGF-ß family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.
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Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Redução de Peso/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Glicosilação , Humanos , Camundongos , Mutação Puntual , Engenharia de ProteínasRESUMO
We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.
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Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Nuclear factor κB (NF-κB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-κB RelA core response.
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Inflamação/genética , Fator de Transcrição RelA/metabolismo , Ativação Transcricional/genética , Animais , Sequência de Bases , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Lógica , Camundongos Endogâmicos C57BL , Modelos Biológicos , Domínios Proteicos , Fator de Necrose Tumoral alfaRESUMO
For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.