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1.
J Mol Biol ; 435(23): 168300, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37805067

RESUMO

Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:ßheterodimer. For human αsubunits it has been shown that they depend on theirßsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same ßsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsßsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its ßsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.


Assuntos
Interleucina-12 , Interleucina-23 , Multimerização Proteica , Humanos , Interleucina-12/química , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-23/química , Interleucina-23/genética , Interleucina-23/metabolismo , Chaperonas Moleculares , Dobramento de Proteína , Mutação , Conformação Proteica , Engenharia de Proteínas , Simulação por Computador
2.
Eur J Pharm Sci ; 190: 106568, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37619953

RESUMO

The cytokine IL-23 activates the IL-23 receptor (IL-23R) and stimulates the differentiation of naïve T helper (Th) cells into a Th17 cell population that secretes inflammatory cytokines and chemokines. This IL-23/Th17 proinflammatory axis drives inflammation in Crohn's disease and ulcerative colitis and represents a therapeutic target of monoclonal antibodies. Non-immunoglobulin binding proteins based on the Streptococcus albumin-binding domain (ABD) provide a small protein alternative to monoclonal antibodies. They can be readily expressed in bacteria. Lactococcus lactis is a safe lactic acid bacterium that has previously been engineered as a vector for the delivery of recombinant therapeutic proteins to mucosal surfaces. Here, L. lactis was engineered to display or secrete ABD-variants against the IL-17 receptor (IL-17R). Its expression and functionality were confirmed with flow cytometry using specific antibody and recombinant IL-17R, respectively. In addition, L. lactis were engineered into multifunctional bacteria that simultaneously express two binders from pNBBX plasmid. First, binders of IL-17R were combined with binder of IL-17. Second, binders of IL-23R were combined with binders of IL-23. The dual functionality of the bacteria was confirmed by flow cytometry using corresponding targets, namely the recombinant receptors IL-17R and IL-23R or the p19 subunit of IL-23. Binding of IL-17 was confirmed by ELISA. With the latter, 97% of IL-17 was removed from solution by 2 × 109 recombinant bacteria. Moreover, multifunctional bacteria targeting IL-17/IL-17R prevented IL-17A-mediated activation of downstream signaling pathways in HEK-Blue IL-17 cell model. Thus, we have developed several multifunctional L. lactis capable of targeting multiple factors of the IL-23/Th17 proinflammatory axis. This represents a novel therapeutic strategy with synergistic potential for the treatment of intestinal inflammations.


Assuntos
Citocinas , Lactococcus lactis , Humanos , Citocinas/metabolismo , Interleucina-17/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fatores Imunológicos , Inflamação , Proteínas de Transporte/metabolismo , Proteínas Recombinantes/metabolismo , Albuminas/metabolismo , Interleucina-23/química , Interleucina-23/metabolismo , Anticorpos Monoclonais
3.
Trends Biochem Sci ; 47(11): 936-949, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35691784

RESUMO

Interleukin 12 (IL-12) family cytokines are secreted proteins that regulate immune responses. Each family member is a heterodimer and nature uses shared building blocks to assemble the functionally distinct IL-12 cytokines. In recent years we have gained insights into the molecular principles and cellular regulation of IL-12 family biogenesis. For each of the family members, generally one subunit depends on its partner to acquire its native structure and be secreted from immune cells. If unpaired, molecular chaperones retain these subunits in cells. This allows cells to regulate and control secretion of the highly potent IL-12 family cytokines. Molecular insights gained into IL-12 family biogenesis, structure, and function now allow us to engineer IL-12 family cytokines to develop novel immunotherapeutic approaches.


Assuntos
Citocinas , Interleucina-12 , Interleucina-12/química , Interleucina-12/metabolismo , Interleucina-23/química , Interleucina-23/metabolismo , Chaperonas Moleculares/metabolismo
5.
Cell Chem Biol ; 29(1): 19-29.e6, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34038748

RESUMO

Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rß1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rß1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Interleucina-23/imunologia , Luciferases/imunologia , Receptores de Interleucina/imunologia , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interleucina-23/química , Luciferases/metabolismo , Ligação Proteica , Receptores de Interleucina/química
6.
Cells ; 9(10)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998371

RESUMO

Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein-protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.


Assuntos
Doenças Autoimunes/genética , Interleucina-12/genética , Interleucina-23/genética , Neoplasias/genética , Sequência de Aminoácidos/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Humanos , Interleucina-12/imunologia , Interleucina-23/química , Interleucina-23/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Ligação Proteica/genética , Transdução de Sinais/genética , Homologia Estrutural de Proteína
7.
J Mol Model ; 26(9): 225, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778954

RESUMO

Rheumatoid arthritis disease is a chronic auto-immune inflammatory disease that mainly causes synovial joint inflammation and cartilage destruction. The tumor necrosis factor-α (TNF-α) is a pivotal cytokine that plays an important role in rheumatoid arthritis. The treatments focusing on a single cytokine inhibition are clinically able to produce meaningful responses in only about half of the treated patients due to multiple cytokines involved in this disease. In the present study, a bispecific tandem single-chain variable fragment was designed in order to suppress both human tumor necrosis factor-α and interleukin-23 (IL23) as a potential therapeutic drug candidate for this disease. To do so, at first, eight bispecific tandem single-chain variable fragment models were built against tumor necrosis factor-α and interleukin-23 cytokines with different domain orders by the homology modeling, and then 50 ns molecular dynamics simulation was performed for each one and then structural properties were exploited. The MD simulation results indicate the fact that the domains' order strongly affects tandem single-chain variable fragment properties, and in overall, the fragment VLAIL23+Linker+VHAIL23+linker+VLATNF+Linker +VHATNF +His6 (VL and VH are light and heavy chain variable fragments and AIL23 and ATNF are anti-interleukin 23 and anti-tumor necrosis factor-α, respectively, and His6 is the six histidine) not only separated antibody domains accurately but also had better stability and solvation free energy. Therefore, this structure can be considered as an effective potential drug for rheumatoid arthritis. It is expected that the findings of this research could shed a light on the treatment approaches of the rheumatoid arthritis disease.


Assuntos
Anticorpos Biespecíficos/química , Interleucina-23/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Anticorpos de Cadeia Única/química , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Anticorpos Biespecíficos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Interleucina-23/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Anticorpos de Cadeia Única/farmacologia , Solventes , Eletricidade Estática , Fator de Necrose Tumoral alfa/antagonistas & inibidores
8.
Adv Protein Chem Struct Biol ; 121: 253-303, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32312425

RESUMO

Protein-protein interactions are central to biology and provide opportunities to modulate disease with small-molecule or protein therapeutics. Recent developments in the understanding of the tractability of protein-protein interactions are discussed with a focus on the ligandable nature of protein-protein interaction surfaces. General principles of inhibiting protein-protein interactions are illustrated with structural biology examples from six members of the IL-23/IL-17 signaling family (IL-1, IL-6, IL-17, IL-23 RORγT and TNFα). These examples illustrate the different approaches to discover protein-protein interaction inhibitors on a target-specific basis that has proven fruitful in terms of discovering both small molecule and biologic based protein-protein interaction inhibitors.


Assuntos
Artrite/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Interleucina-17/antagonistas & inibidores , Interleucina-23/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Artrite/genética , Artrite/imunologia , Artrite/patologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Sítios de Ligação/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Fatores Imunológicos/química , Interleucina-17/química , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-23/química , Interleucina-23/genética , Interleucina-23/imunologia , Modelos Moleculares , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
9.
Sci Rep ; 9(1): 16850, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727948

RESUMO

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Antígenos de Histocompatibilidade Classe I/genética , Interleucina-23/genética , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Análise Fatorial , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Corpos de Inclusão/química , Interleucina-23/química , Interleucina-23/isolamento & purificação , Isopropiltiogalactosídeo/farmacologia , Análise de Componente Principal , Redobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Solubilidade
10.
Nat Commun ; 10(1): 4121, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511508

RESUMO

The functionality of most secreted proteins depends on their assembly into a defined quaternary structure. Despite this, it remains unclear how cells discriminate unassembled proteins en route to the native state from misfolded ones that need to be degraded. Here we show how chaperones can regulate and control assembly of heterodimeric proteins, using interleukin 23 (IL-23) as a model. We find that the IL-23 α-subunit remains partially unstructured until assembly with its ß-subunit occurs and identify a major site of incomplete folding. Incomplete folding is recognized by different chaperones along the secretory pathway, realizing reliable assembly control by sequential checkpoints. Structural optimization of the chaperone recognition site allows it to bypass quality control checkpoints and provides a secretion-competent IL-23α subunit, which can still form functional heterodimeric IL-23. Thus, locally-restricted incomplete folding within single-domain proteins can be used to regulate and control their assembly.


Assuntos
Interleucina-23/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Meia-Vida , Humanos , Interleucina-23/química , Modelos Biológicos , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína
11.
Biochem Pharmacol ; 165: 240-248, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30885765

RESUMO

The four core members of the Interleukin-12 (IL-12) family of cytokines, IL-12, IL-23, IL-27 and IL-35 are heterodimers which share α- and ß-cytokine subunits. All four cytokines are immune modulators and have been proposed to play divergent roles in inflammatory arthritis. In recent years additional combinations of α- and ß-cytokine subunits belonging to the IL-12 family have been proposed to form novel cytokines such as IL-39. However, the actual extent of the combinatorial potential of the cytokine subunits in the human IL-12 family is not known. Here, we identify several combinations of subunits that form secreted heterodimeric assemblies based on a systematic orthogonal approach. The heterodimers are detected in the conditioned media harvested from mammalian cell cultures transfected with unfused pairs of cytokine subunits. While certain previously reported subunit combinations could not be recapitulated, our approach showed robustly that all four of the canonical members could be secreted. Furthermore, we provide evidence for the interaction between Cytokine Receptor Like Factor 1 (CRLF1) and Interleukin-12 subunit alpha (p35). Similar to IL-27 and IL-35 this novel heterodimer is not abundantly secreted rendering isolation from the conditioned medium very challenging, unlike IL-12 and IL-23. Our findings set the stage for fine-tuning approaches towards the biochemical reconstitution of IL-12 family cytokines for biochemical, cellular, and structural studies.


Assuntos
Interleucina-12/química , Interleucina-23/biossíntese , Interleucinas/química , Proteínas Recombinantes de Fusão/química , Células HEK293 , Humanos , Interleucina-12/biossíntese , Interleucina-23/química , Interleucinas/biossíntese , Multimerização Proteica , Subunidades Proteicas/biossíntese , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão/biossíntese
12.
J Recept Signal Transduct Res ; 38(4): 327-334, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30481093

RESUMO

Wealth of structural data on theurapeutic targets in complex with monoclonal antibodies (mAbs) and advances in molecular modeling algorithms present exciting opportunities in the field of novel biologic design. Interleukin 23 (IL23), a well-known drug target for autoimmune diseases, in complex with mAb 7G10 offers prospect to design potent lead antibodies by traversing the complete epitope-paratope interface. Herein, key interactions aiding antibody-based neutralization in IL23-7G10 complex are resolute through PyMOL, LigPlot+, Antibody i-Patch, DiscoTope and FoldX. Six amino acids Ser31, Val33, Asn55, Lys59 in heavy chain and His34, Ser93 in light chain are subjected to in silico mutagenesis with residues Met, Trp, Ile, Leu and Arg. A set of 431 mutant macromolecules are outlined. Binding affinities of these molecules with IL23 are estimated through protein-protein docking by employing ZDOCK, ClusPro and RosettaDock. Subsequently, the macromolecules revealed comparable result with 7G10 are cross validated through binding free-energy calculations by applying Molecular Mechanics/Poisson Boltzman Surface Area method in CHARMM. Thirty nine designed theoretical antibodies showed improved outcome in all evaluations; from these, top 10 molecules showed at least nine unit better binding affinity compared to the known mAb. These molecules have the potential to act as lead antibodies. Subsequent molecular dynamics simulations too favored prospective of best ranked molecule to have therapeutic implications in autoimmune and inflammatory diseases. Abbreviations: IL23: interleukin 23; IL17: interleukin17; Ab: antibody; Ag: antigen; mAbs: monoclonal antibodies; STAT3: signal transducer and activator of transcription 3; STAT4: signal transducer and activator of transcription 4; PDB: protein databank; MM/PBSA: molecular mechanics Poisson-Boltzmann surface area; Ag-Ab: antigen- antibody complex; SPC/E: extended simple point charge; SD: steepest descents; PME: particle mesh ewald; dG: binding free energies; Fv: variable fragment.


Assuntos
Anticorpos Monoclonais/imunologia , Doenças Autoimunes/imunologia , Inflamação/imunologia , Interleucina-23/química , Anticorpos Monoclonais/química , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Sítios de Ligação de Anticorpos , Biologia Computacional , Epitopos/química , Epitopos/imunologia , Humanos , Ligação de Hidrogênio , Inflamação/tratamento farmacológico , Inflamação/genética , Interleucina-23/imunologia , Simulação de Dinâmica Molecular , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT4/química , Fator de Transcrição STAT4/imunologia
13.
Exp Dermatol ; 27(7): 737-747, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29704872

RESUMO

Immune cells and cytokines play an important role in the pathogenesis of psoriasis. Interleukin-12 (IL-12) and IL-23 promote cellular responses mediated by T cells, which contribute to an inflammatory loop responsible for the induction and maintenance of psoriatic plaques. Antibodies that inhibit IL-12/23 or IL-23 are key treatment options for patients with psoriasis. IL-12 and IL-23 also play a key role in immune responses to infections and tumors. A growing body of information from clinical trials, cohort studies, postmarketing reports, genetic studies and animal models provides insights into the potential biological relationships between IL-12/23 inhibition and malignancies. We summarize this information in tables and provide some context for the interpretation of these data with the goal of informing dermatologists who are using IL-12/23 or IL-23 inhibitors to treat patients with psoriasis.


Assuntos
Interleucina-12/antagonistas & inibidores , Interleucina-23/antagonistas & inibidores , Neoplasias/etiologia , Psoríase/imunologia , Psoríase/terapia , Animais , Ensaios Clínicos como Assunto , Fármacos Dermatológicos/efeitos adversos , Fármacos Dermatológicos/uso terapêutico , Modelos Animais de Doenças , Humanos , Imunidade Celular , Interleucina-12/química , Interleucina-12/imunologia , Interleucina-23/química , Interleucina-23/imunologia , Camundongos , Modelos Imunológicos , Vigilância de Produtos Comercializados , Psoríase/complicações , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina-12/deficiência , Receptores de Interleucina-12/genética , Linfócitos T/imunologia , Ustekinumab/efeitos adversos , Ustekinumab/uso terapêutico
14.
Fish Shellfish Immunol ; 71: 434-442, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29024769

RESUMO

Interleukin (IL-) 23, a member of IL-12 family, is a composite cytokine with the subunits of p19 and p40. Although IL-12 and IL-23 share the p40 subunit, they play vastly different roles in immune regulation. In teleost, much emphasis has been placed on the identification of IL-12, but evidence for the existence of IL-23 is still lacking. In the present study, a p19 gene and three p40 paralogues were isolated and identified from grass carp, suggesting multiple assembly of IL-23 molecules in fish species. To address this issue, the existence of different p19/p40 heterodimers were examined by Co-Immunoprecipitation (Co-IP) assay, showing that only co-expression of p19 and each p40 subunit could produce the soluble proteins corresponding to three IL-23 isoforms. Additionally, bacterial infection could up-regulate the mRNA expression of p19, p40a and p40b but not p40c in head kidney, indicating distinct expression patterns of three p40 paralogues. Moreover, in vitro experiments demonstrated that both B-cell stimulator, LPS and T-cell mitogen, PHA markedly increased the mRNA levels of p19 and three p40 paralogues in grass carp periphery blood lymphocytes (PBLs). The simultaneous up-regulation of mRNA expression of p19 and p40 paralogues in response to immune stimuli supports the idea that p19 may form heterodimeric molecules with three p40 subunits in grass carp under immune activation. These findings for the first time highlight the potential of p19 and p40 for dimerization in fish, particularly the existence of three IL-23 isoforms as soluble heterodimeric cytokines in grass carp, thereby providing the basis for further investigating the function of IL-23 in fish immunity.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-23/genética , Interleucina-23/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Interleucina-23/química , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Alinhamento de Sequência/veterinária
15.
Autoimmunity ; 50(2): 102-113, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28100093

RESUMO

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.


Assuntos
Subunidade p19 da Interleucina-23/metabolismo , Interleucina-23/metabolismo , Ativação Linfocitária/imunologia , Células Th17/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Interleucina-23/química , Subunidade p19 da Interleucina-23/química , Subunidade p19 da Interleucina-23/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Modelos Moleculares , Fagócitos/imunologia , Fagócitos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Interleucina/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/imunologia
16.
PLoS One ; 10(11): e0142930, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26566272

RESUMO

BACKGROUND: Immunogenicity due to antidrug antibodies (ADA) to tumor necrosis factor (TNF)-α antagonists is known to decrease treatment response. However, few studies have investigated ADA in ustekinumab, an interleukin-12 and -23 antagonist, in a clinical setting. This study aimed to investigate the immunogenicity of ustekinumab and its clinical consequences in psoriasis. METHODS: This prospective observational study enrolled 76 patients with plaque psoriasis who were treated with ustekinumab for a minimum of 7 months. Blood samples were drawn just prior to scheduled ustekinumab injection during clinic visits. Levels of anti-ustekinumab antibody (AUA) and serum ustekinumab concentration were measured respectively by radioimmunoassays and enzyme-linked immunoassays respectively, and correlated to clinical data and Psoriasis Area and Severity Index (PASI). RESULTS: AUA was detected in 6.5% of patients after a mean of 13 months of treatment. Patients with positive AUA had significantly lower serum ustekinumab concentrations (0.01 vs. 0.2 mg/L, p<0.001) and lower PASI 50 response than patients without AUA (0% vs. 69%, p = 0.004).The percentage of AUA formation was comparable between patients who had failed previous adalimumab with or without anti-adalimumab antibodies (AAA) (14.3% vs. 12.5%, p = 1.00). However, a higher proportion of switchers without AAA obtaining PASI50 (71.4% vs. 37.5%) and PASI75 response (42.9% vs.12.5%) within 7 months of ustekinumab treatment than with AAA though this difference did not reach statistical significance. CONCLUSIONS: Our results suggest that presence of AUA was significantly associated with treatment failure for ustekinumab, though limited by a small sample size. Also, determining the presence of ADA to antecedent TNF-α antagonists may assist in choosing an optimized subsequent treatment modality achieving treatment success.


Assuntos
Adalimumab/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Psoríase/tratamento farmacológico , Psoríase/imunologia , Ustekinumab/uso terapêutico , Adalimumab/administração & dosagem , Adulto , Anticorpos/sangue , Fármacos Dermatológicos/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/tratamento farmacológico , Interleucina-12/química , Interleucina-23/química , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Radioimunoensaio , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Ustekinumab/administração & dosagem
17.
Nature ; 521(7551): 222-6, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-25754330

RESUMO

Psoriasis is a chronic inflammatory skin disorder that affects approximately 2-3% of the population worldwide and has severe effects on patients' physical and psychological well-being. The discovery that psoriasis is an immune-mediated disease has led to more targeted, effective therapies; recent advances have focused on the interleukin (IL)-12/23p40 subunit shared by IL-12 and IL-23. Evidence suggests that specific inhibition of IL-23 would result in improvement in psoriasis. Here we evaluate tildrakizumab, a monoclonal antibody that targets the IL-23p19 subunit, in a three-part, randomized, placebo-controlled, sequential, rising multiple-dose phase I study in patients with moderate-to-severe psoriasis to provide clinical proof that specific targeting of IL-23p19 results in symptomatic improvement of disease severity in human subjects. A 75% reduction in the psoriasis area and severity index (PASI) score (PASI75) was achieved by all subjects in parts 1 and 3 (pooled) in the 3 and 10 mg kg(-1) groups by day 196. In part 2, 10 out of 15 subjects in the 3 mg kg(-1) group and 13 out of 14 subjects in the 10 mg kg(-1) group achieved a PASI75 by day 112. Tildrakizumab demonstrated important clinical improvement in moderate-to-severe psoriasis patients as demonstrated by improvements in PASI scores and histological samples.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia , Interleucina-23/antagonistas & inibidores , Terapia de Alvo Molecular , Psoríase/tratamento farmacológico , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Método Duplo-Cego , Epitélio/efeitos dos fármacos , Epitélio/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-23/química , Interleucina-23/imunologia , Pessoa de Meia-Idade , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/patologia , Resultado do Tratamento , Adulto Jovem
18.
Expert Rev Proteomics ; 12(2): 159-69, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25711416

RESUMO

IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.


Assuntos
Terapia Biológica , Deutério/química , Hidrogênio/química , Interleucina-23/química , Biologia Computacional , Humanos , Interleucina-23/metabolismo , Espectrometria de Massas/métodos , Ligação Proteica , Conformação Proteica , Receptores de Interleucina/química
19.
Cytokine ; 71(2): 348-59, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25516297

RESUMO

Human IL12RB1 encodes IL12Rß1, a type I transmembrane receptor that is an essential component of the IL12- and IL23-signaling complex. IL12RB1 is well-established as being a promoter of delayed type hypersensitivity (DTH), the immunological reaction that limits tuberculosis. However, recent data demonstrate that in addition to promoting DTH, IL12RB1 also promotes autoimmunity. The contradictory roles of IL12RB1 in human health raises the question, what are the factors governing IL12RB1 function in a given individual, and how is inter-individual variability in IL12RB1 function introduced? Here we review recent data that demonstrate individual variability in IL12RB1 function is introduced at the epigenetic, genomic polymorphism, and mRNA splicing levels. Where and how these differences contribute to disease susceptibility and outcome are also reviewed. Collectively, recent data support a model wherein IL12RB1 sequence variability - whether introduced at the genomic or post-transcriptional level - contributes to disease, and that human IL12RB1 is not as simple a gene as we once believed.


Assuntos
Subunidade beta 1 de Receptor de Interleucina-12/fisiologia , Processamento Alternativo , Animais , Asma/imunologia , Infecções Bacterianas/imunologia , Epigênese Genética , Éxons , Genômica , Humanos , Interleucina-12/química , Interleucina-23/química , Camundongos , Modelos Genéticos , Neoplasias/imunologia , Filogenia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Dermatopatias/imunologia
20.
J Biol Chem ; 290(1): 359-70, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25371211

RESUMO

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor ß1 (IL-12Rß1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rß1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rß1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rß1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rß1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rß1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rß1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.


Assuntos
Subunidade beta 1 de Receptor de Interleucina-12/química , Subunidade p40 da Interleucina-12/química , Interleucina-23/química , Receptores de Interleucina-12/química , Receptores de Interleucina/química , Animais , Sítios de Ligação , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Expressão Gênica , Humanos , Subunidade beta 1 de Receptor de Interleucina-12/genética , Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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