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1.
MAbs ; 2(3): 335-46, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20424514

RESUMO

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Lúpus Eritematoso Sistêmico/terapia , Receptores de Interleucina-21/antagonistas & inibidores , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Interleucinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-21/imunologia
2.
J Mol Biol ; 382(5): 1168-83, 2008 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-18675824

RESUMO

Interleukin (IL) 22 is a type II cytokine that is produced by immune cells and acts on nonimmune cells to regulate local tissue inflammation. As a product of the recently identified T helper 17 lineage of CD4(+) effector lymphocytes, IL-22 plays a critical role in mucosal immunity as well as in dysregulated inflammation observed in autoimmune diseases. We used comprehensive mutagenesis combined with mammalian cell expression, ELISA cell-based, and structural methods to evaluate how IL-22 interacts with its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22 binding protein. This study identifies those amino acid side chains of IL-22 that are individually important for optimal binding to IL-22R, considerably expands the definition of IL-22 surface required for binding to IL-10R2, and demonstrates how IL-22 binding protein prevents IL-22R from binding to IL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacent IL-22 surfaces contributed mostly by helices A, D, and F and loop AB. Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26 which are other IL-10-like cytokines, interact with their respective cell surface receptors.


Assuntos
Subunidade beta de Receptor de Interleucina-10/química , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucinas/química , Interleucinas/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Linhagem Celular , Humanos , Técnicas In Vitro , Subunidade beta de Receptor de Interleucina-10/genética , Interleucinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Receptores de Interleucina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Termodinâmica , Interleucina 22
3.
J Immunol ; 181(4): 2799-805, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18684971

RESUMO

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.


Assuntos
Interleucina-17/fisiologia , Receptores de Interleucina-17/fisiologia , Receptores de Interleucina/fisiologia , Transdução de Sinais/imunologia , Linhagem Celular , Dimerização , Relação Dose-Resposta Imunológica , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Interleucina-17/metabolismo , Ligação Proteica/imunologia , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/metabolismo
4.
Proc Natl Acad Sci U S A ; 105(1): 33-8, 2008 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18162540

RESUMO

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.


Assuntos
Canais de Cálcio/química , Sirolimo/química , Proteínas de Ligação a Tacrolimo/química , Animais , Cálcio/metabolismo , Eletrofisiologia/métodos , Humanos , Imunofilinas/metabolismo , Imunossupressores/farmacologia , Ligantes , Modelos Químicos , Neuritos/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Acidente Vascular Cerebral/metabolismo
5.
J Immunol ; 179(11): 7791-9, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18025225

RESUMO

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4(+) T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.


Assuntos
Interleucina-17/biossíntese , Pulmão/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva/métodos , Animais , Dimerização , Modelos Animais de Doenças , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos
6.
J Biol Chem ; 282(18): 13447-55, 2007 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-17355969

RESUMO

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-17/imunologia , Ativação Linfocitária/imunologia , Motivos de Aminoácidos , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Cricetinae , Cricetulus , Cisteína/genética , Cisteína/imunologia , Dimerização , Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Imunidade Celular , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-17/farmacologia , Ativação Linfocitária/efeitos dos fármacos
7.
J Immunol ; 170(2): 711-8, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12517932

RESUMO

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4(+) T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Ralpha expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.


Assuntos
Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Superfície/fisiologia , Antígeno B7-1 , Antígenos CD28/biossíntese , Citocinas/fisiologia , Interleucina-15/biossíntese , Interleucina-4/biossíntese , Interleucina-7/biossíntese , Interleucinas/biossíntese , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Anticorpos/fisiologia , Antígenos CD , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose , Antígeno B7-H1 , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/fisiologia , Divisão Celular/imunologia , Células Cultivadas , Regulação para Baixo/imunologia , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Peptídeos e Proteínas de Sinalização Intercelular , Subunidade gama Comum de Receptores de Interleucina , Interleucina-2/fisiologia , Interleucina-7/fisiologia , Glicoproteínas de Membrana , Peptídeos/fisiologia , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina-7/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
8.
Curr Protoc Immunol ; Chapter 6: 6.15.1-6.15.12, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18432806

RESUMO

This unit describes an ELISA and a cell proliferation assay that can be used, respectively, to measure the protein level or biologic activity of human and murine interleukin 11 (IL-11). The bioassay is based on the ability of IL-11 to support growth of the B9-11 cell line, a subline of B9 that has traditionally been used to measure levels of IL-6. B9-11 is substantially more responsive to IL-11 than the T10 line used in older protocols. This new bioassay therefore provides improved sensitivity, with a detection limit of 20 pg/ml. An alternate procedure is provided that employs neutralizing antibodies in the cell proliferation bioassay to use to ensure that the activity of the desired molecule (IL-11) is being measured in samples containing multiple cytokines. A describes maintenance of B9-11 cells.


Assuntos
Bioensaio/métodos , Proliferação de Células , Ensaio de Imunoadsorção Enzimática/métodos , Interleucina-11/análise , Anticorpos Monoclonais , Linhagem Celular , Humanos , Hidroquinonas , Interleucina-11/imunologia , Interleucina-11/fisiologia , Sensibilidade e Especificidade
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