RESUMO
There are currently two Food and Drug Administration-approved classes of biologic agents that target tumor necrosis factor-alpha (TNF-alpha): anti-TNF monoclonal antibodies (mAbs) (adalimumab and infliximab), and soluble TNF receptors (etanercept). This study examined the ability of the TNF antagonists to: (1) bind various polymorphic variants of cell surface-expressed Fc receptors (FcgammaRs) and the complement component C1q, and (2) mediate Ab-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) killing of cells expressing membrane-bound TNF (mTNF) in vitro. Both mAbs and the soluble TNF receptor demonstrated low-level binding to the activating receptors FcgammaRI, FcgammaRIIa, and FcgammaRIIIa, and the inhibitory receptor FcgammaRIIb, in the absence of exogenous TNF. However, upon addition of TNF, the mAbs, but not etanercept, showed significantly increased binding, in particular to the FcgammaRII and FcgammaRIII receptors. Infliximab and adalimumab induced ADCC much more potently than etanercept. In the presence of TNF, both mAbs bound C1q in in vitro assays, but etanercept did not bind C1q under any conditions. Infliximab and adalimumab also induced CDC in cells expressing mTNF more potently than etanercept. Differences in the ability to bind ligand and mediate cell death may account for the differences in efficacy and safety of TNF antagonists.
Assuntos
Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa , Adalimumab , Anticorpos Monoclonais Humanizados , Linhagem Celular , Complemento C1q/metabolismo , Citotoxicidade Imunológica , Etanercepte , Humanos , Infliximab , Receptores de IgG/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/química , Naftalenossulfonato de Anilina , Dicroísmo Circular , Corantes Fluorescentes , Humanos , Indicadores e Reagentes , Proteína Antagonista do Receptor de Interleucina 1/genética , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Desnaturação Proteica , Dobramento de Proteína , Termodinâmica , Triptofano/química , UreiaRESUMO
A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E. coli. This soluble receptor contains the first 2.6 of the 4 domains of the intact sTNF-RI molecule. A monoPEGylated form of this molecule was produced using a 30 kD methoxyPEG aldehyde with approximately 85% selectivity for the N-terminal amino group. This molecule was shown to be less immunogenic in primates than the full length (4.0 domain) molecule or other versions of sTNF-RI which were either PEGylated at different sites or with different molecular weight PEGs. The 30 kD PEG also has a longer serum half-life to the molecule than lower molecular weight PEGs. This molecule markedly blunts the inflammatory response in a number of rheumatoid arthritis animal models. In addition, phase I/II and early phase II data in humans indicate that PEG sTNF-RI is non-immunogenic and that weekly dosing with this drug can reduce the number of tender and swollen joints in rheumatoid arthritis patients. PEG sTNF-RI has comparable American College of Rheumatology (ACR) efficacy scores as other anti-TNF molecules currently used to treat rheumatoid arthritic patients.
Assuntos
Antígenos CD , Artrite Reumatoide/tratamento farmacológico , Receptores do Fator de Necrose Tumoral , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/uso terapêutico , Área Sob a Curva , Disponibilidade Biológica , Modelos Animais de Doenças , Feminino , Meia-Vida , Humanos , Inflamação/tratamento farmacológico , Masculino , Taxa de Depuração Metabólica , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/uso terapêutico , Receptores Tipo I de Fatores de Necrose Tumoral , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
UNLABELLED: Conatumumab is a fully human monoclonal antibody that binds to and activates human death receptor 5 (DR5; also known as TRAIL receptor 2). The purpose of this study was to characterize (64)Cu-labeled conatumumab as a PET tracer for imaging DR5 in tumors. METHODS: DOTA-conatumumab was synthesized by incubating conatumumab with 2,2',2â³-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-NHS). The absolute numbers of DOTA molecules per conatumumab molecules were determined by matrix-assisted laser desorption ionization mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. (64)Cu-DOTA-conatumumab was prepared by incubating (64)CuCl(2) (33-222 MBq) with DOTA-conatumumab at 37°C for 1 h. Binding of conatumumab and DOTA-conatumumab to Fc-coupled human DR5 (huTR2-Fc) was tested in a kinetic analysis assay, and the biologic activity of copper-DOTA-conatumumab was measured using a caspase-3/7 luminescent assay. In vivo evaluation of DOTA-conatumumab and copper-DOTA-conatumumab was done in severe combined immunodeficiency mice bearing Colo205 xenografts: tissue uptake was determined with biodistribution studies, and small-animal PET and autoradiography were used to determine the uptake of (64)Cu-DOTA conatumumab into tumors and other tissues. RESULTS: DOTA-conatumumab was prepared with an average of 5 DOTA molecules per conatumumab molecule. The in vitro median effective concentration required to induce a 50% effect of DOTA-conatumumab and conatumumab from the assay were 389 and 320 pM, respectively. The median effective dose (±SD) of DOTA-conatumumab and conatumumab via the caspase assay was 135 ± 31 and 128 ± 30 pM, respectively. In female CB17 severe combined immunodeficiency mice bearing Colo205 xenografts, DOTA-conatumumab and conatumumab inhibited tumor growth to the same extent. Small-animal PET studies showed tumor uptake at 24 h after injection of the tracer, with a mean standardized uptake value of 3.16 (n = 2). Tumor uptake was decreased by the coadministration of 400 µg of unlabeled conatumumab (mean standardized uptake value, 1.55; n = 2), suggesting saturable uptake. Tissue uptake determined by biodistribution studies was in agreement with the small-animal PET findings. CONCLUSION: These results suggest that (64)Cu-DOTA-conatumumab is a potential PET tracer for imaging DR5 in tumors and may be useful for measuring on-target occupancy by conatumumab.
Assuntos
Anticorpos Monoclonais , Compostos Organometálicos , Compostos Radiofarmacêuticos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Autorradiografia , Caspase 3/metabolismo , Caspase 7/metabolismo , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Marcação por Isótopo/métodos , Camundongos , Camundongos SCID , Neoplasias/diagnóstico por imagem , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição TecidualRESUMO
Tumor necrosis factor (TNF) antagonists are efficacious in the treatment of various autoimmune diseases. Two classes of TNF antagonists are currently commercially available: soluble TNF receptor-Fc fusion proteins (etanercept) and anti-TNF mAbs (adalimumab and infliximab). The classes differ in molecular structures and mechanisms of action. The interactions between TNF antagonists with TNF molecules were characterized. The anti-TNF mAbs, but not the soluble TNF receptor, formed visible lines of precipitation in Ouchterlony assays. The molecular weights of complexes formed by TNF (52 kDa) with either etanercept (130 kDa), adalimumab (150 kDa), or infliximab (average 165 kDa) were determined by size exclusion chromatography-light-scattering assays. Etanercept and TNF formed complexes of 180 and 300 kDa, representing one and two etanercept monomers bound to a TNF trimer, respectively. Adalimumab and infliximab formed a variety of complexes with TNF with molecular weights as high as 4,000 and 14,000 kDa, respectively, suggesting the presence of complexes with a wide range of sizes and stoichiometries. The absence of large complex formation with the binding of soluble receptor-fusion proteins to TNF may account for the different clinical efficacy and safety profiles of the two classes of TNF antagonists.
Assuntos
Anticorpos Monoclonais/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Animais , Anticorpos Monoclonais Humanizados , Etanercepte , Humanos , Imunoglobulina G/metabolismo , Técnicas In Vitro , Infliximab , Cinética , Complexos Multiproteicos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Etanercept (Amgen Inc, Thousand Oaks, CA) is a human soluble p75 tumor necrosis factor (TNF) receptor-human-IgG1 (hup75 TNFR-huIgG1) fusion protein used in the treatment of chronic inflammatory diseases in humans, including rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. To be able to study the effects of the soluble receptor fusion protein in mouse models, including those that mimic human granulomatous infections, a murine soluble p75-TNF receptor-murine IgG1 (murine p75-murine IgG1) fusion protein had to be constructed. This article discusses the generation, large-scale production, and purification of this molecule.
Assuntos
Imunoglobulina G/biossíntese , Imunoglobulina G/isolamento & purificação , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Animais , Etanercepte , Humanos , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Especificidade da EspécieRESUMO
Overproduction of inflammatory mediators, such as tumor necrosis factor (TNF), is key to the development and maintenance of inflammatory processes. Etanercept is a soluble TNF receptor fusion protein used in the treatment of various chronic inflammatory diseases, including rheumatoid arthritis and psoriasis. This study investigated the effects of murine p75-Fc, a soluble TNF receptor protein, on TNF-induced IL-6 production in mice. Six groups of mice received either murine p75-Fc (0.15, 0.50, 1.5, 5, and 15 mg/kg) or phosphate-buffered saline. Three days later, mice were injected intravenously with 10 microg of murine TNF and blood samples were taken after 3 hours. Serum IL-6 and TNF were measured by ELISA. Mice treated with 5 and 15 mg/kg murine p75-Fc demonstrated complete inhibition of TNF-induced IL-6 production. Murine p75-Fc (1.5 mg/kg) resulted in a partial but significant reduction of TNF-induced IL-6 production. No TNF was detected in 5 and 15 mg/kg murine p75-Fc-treated mice, except one in the 5 mg/kg dose group. In conclusion, murine p75-Fc completely inhibits TNF-induced IL-6 production in mice.
Assuntos
Imunoglobulina G/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Etanercepte , Humanos , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Tumor necrosis factor (TNF) plays an essential role in the immunologic maintenance of Mycobacterium tuberculosis infection. Although an increased rate of tuberculosis has been reported in humans treated with anti-TNF biological agents, disparate rates of disease have been observed between those treated with infliximab, an anti-TNF antibody, and etanercept, a TNF-neutralizing TNF receptor (TNFR) fusion molecule. We compared the effects of anti-TNF antibody and soluble TNFR fusion molecule in the murine model of tuberculosis. Systemic TNF neutralization was equivalent between these molecules, and both resulted in rapid morbidity at the initiation of infection. During chronic infection, administration of the receptor fusion molecule allowed the control of infection, whereas antibody treatment caused mice to die within a month. We provide evidence of decreased penetration into the granulomas by the receptor fusion molecule, compared with antibody. These findings begin to clarify the mechanistic difference between anti-TNF agents and their role in the exacerbation of tuberculosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoglobulina G/imunologia , Pulmão/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Tuberculose Pulmonar , Fator de Necrose Tumoral alfa/imunologia , Doença Aguda , Animais , Anticorpos Monoclonais/imunologia , Doença Crônica , Complemento C3/deficiência , Complemento C3/metabolismo , Etanercepte , Feminino , Humanos , Imunoglobulina G/uso terapêutico , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Proteínas Recombinantes de Fusão/imunologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/fisiopatologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.
Assuntos
Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/metabolismo , Marcadores de Afinidade/metabolismo , Ânions/metabolismo , Sítios de Ligação/genética , Ligação Competitiva/genética , Cátions/metabolismo , Ácido Cítrico/metabolismo , Difosfatos/metabolismo , Temperatura Alta , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Lisina/genética , Mutagênese Sítio-Dirigida , Fosfatos/metabolismo , Ácido Fosfonoacéticos/análogos & derivados , Ácido Fosfonoacéticos/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Sialoglicoproteínas/antagonistas & inibidores , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Transdução de Sinais/genética , Relação Estrutura-AtividadeRESUMO
We have characterized a receptor:ligand pair, ICOS:B7RP-1, that is structurally and functionally related to CD28:B7.1/2. We reported previously that B7RP-1 costimulates T cell proliferation and immune responses (Yoshinaga et al., Nature 1999;402:827-32; Guo et al., J Immunol 2001;166:5578-84; Yoshinaga et al., Int Immunol 2000;12:1439-47). We report that B7RP-1-Fc causes rejection or growth inhibition of Meth A, SA-1 and EMT6 tumors in syngeneic mice. Established Meth A tumors were rejected effectively with a single dose of B7RP-1-Fc, however, the treatment was less effective on larger tumors. Mice that rejected Meth A tumors previously by Day 30, also rejected a subsequent Meth A challenge on Day 60, without additional B7RP-1-Fc treatment, indicating a long-lived memory response. Tumor cells believed to be less immunogenic, such as P815 and EL-4 cells, were less responsive to this treatment. The EL-4 responsiveness to the B7RP-1-Fc treatment was enhanced, however, by pre-treatment of the mice with cyclophosphamide. As expected, T cells appeared to be targeted by B7RP-1-Fc treatment. Thus, the administration of soluble B7RP-1-Fc may have therapeutic value in generating or enhancing anti-tumor activity in a clinical setting.