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1.
Mol Immunol ; 170: 19-25, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38598870

RESUMO

The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.


Assuntos
Anticorpos Monoclonais , Complemento C6 , Hemólise , Animais , Humanos , Ratos , Cobaias , Camundongos , Hemólise/efeitos dos fármacos , Hemólise/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Complemento C6/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/imunologia , Ativação do Complemento/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Reações Cruzadas/imunologia
2.
J Innate Immun ; 16(1): 56-65, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38035563

RESUMO

INTRODUCTION: C3 is central for all complement activation pathways, thus making it an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for clinical use. However, most, if not all, C3 inhibitors are human or nonhuman primate C3-specific, making evaluating their efficacies in vivo before a clinical trial extremely difficult and costly. METHODS: We first studied the compatibility of human C3 in the rat complement system, then developed a C3 humanized rat using the CRISPR/Cas9 technology. We thoroughly characterized the resultant human C3 humanized rats and tested the treatment efficacy of an established primate-specific C3 inhibitor in a model of complement-mediated hemolysis in the C3 humanized rats. RESULTS: We found that supplementing human C3 protein into the C3-deficient rat blood restored its complement activity, which was inhibited by rat factor H or compstatin, suggesting that human C3 is compatible to the rat complement system. The newly developed C3 humanized rats appeared healthy and expressed human but not rat C3 without detectable spontaneous C3 activation. More importantly, complement-mediated hemolysis in the C3 humanized rats was also inhibited by compstatin both in vitro and in vivo. CONCLUSION: The successfully developed C3 humanized rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.


Assuntos
Ativação do Complemento , Hemólise , Ratos , Humanos , Animais , Primatas
3.
Clin Immunol ; 253: 109678, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37315680

RESUMO

C2 is an attractive therapeutic target for many complement-mediated diseases. We developed Nab1B10, a new anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation. Mechanistically, Nab1B10 binds to the C2a portion of C2 and inhibits the assembly of C3 convertase C4b2a. Nab1B10 cross-reacts with monkey but not rodent C2 and inhibits classical pathway-mediated hemolysis. Using a new complement humanized mouse model of autoimmune hemolytic anemia (AIHA), we demonstrated that Nab1B10 abolished classical pathway complement activation-mediated hemolysis in vivo. We also developed C2-neutralizing bi- and tetra-valent antibodies based on Nab1B10 and found these antibodies significantly more potent than the other anti-C2 monoclonal antibody that is already in clinical trials. These data suggest that these novel C2-neutralizing nanobodies could be further developed as new therapeutics for many complement-mediated diseases, in which pathogenesis is dependent on the classical and/or lectin pathway of complement activation.


Assuntos
Anemia Hemolítica Autoimune , Complemento C2 , Camundongos , Animais , Complemento C2/metabolismo , Hemólise , Ativação do Complemento , Inativadores do Complemento
4.
Blood Adv ; 4(9): 2049-2057, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32396613

RESUMO

Membrane attack complexes (MACs; C5b-9) assembled after complement activation can directly injure self-tissues, leading to various diseases. Eculizumab, a monoclonal antibody (mAb) against complement component C5, is being used in the clinic to treat diseases in which MAC-mediated tissue damage is a primary cause. However, C5 is not a selective target for MAC assembly inhibition, and some patients respond incompletely or not at all to the eculizumab treatment. Therefore, C6, the next essential component in the terminal pathway of complement activation, may be an alternative target for the selective inhibition of MAC formation. Surprisingly, few reports describe a functional blockade of C6 using a specific mAb. Here, we report the development of an anti-human C6 mAb (clone 1C9) that recognizes C6 both in free circulation and within C5b6 complexes. This mAb blocked C7 binding to C5b6 complexes and consequently inhibited MAC formation and protected affected paroxysmal nocturnal hemoglobinuria patient red blood cells from MAC-mediated damage in vitro. In addition, this mAb cross-reacts with rhesus monkey but not mouse complement C6. Finally, 1C9 significantly reduced human complement-mediated intravascular hemolysis in vivo in a mouse model. These results suggest that the anti-C6 mAb holds promise as a new therapeutic agent that selectively targets MAC for many complement-mediated pathological conditions.


Assuntos
Complemento C6 , Complexo de Ataque à Membrana do Sistema Complemento , Animais , Ativação do Complemento , Complemento C5 , Hemólise , Humanos , Camundongos
5.
Proc Natl Acad Sci U S A ; 110(33): 13510-5, 2013 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-23904479

RESUMO

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1ß in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.


Assuntos
Proteínas F-Box/metabolismo , Regulação da Expressão Gênica/imunologia , Lisina/metabolismo , NF-kappa B/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Citocinas/farmacologia , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Interleucina-1beta/farmacologia , Histona Desmetilases com o Domínio Jumonji , Metilação , Análise em Microsséries , Mutagênese Sítio-Dirigida , Mutação/genética , NF-kappa B/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
6.
J Interferon Cytokine Res ; 30(9): 677-81, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20629536

RESUMO

Transforming growth factor ß2 (TGFß2) is highly expressed in a variety of different cancer cell lines. Using Z12 cells, a mutant of 293 cells with overexpression of TGFß2, we found that the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) sequence in the promoter of the TGFß2 gene is crucial for its increased expression. Further, constitutive phosphorylation of CRE-binding protein (CREB) is increased in these cells. Treating Z12 cells with either the PI3 kinase inhibitor LY294002 or the p38 MAPK inhibitor SB203580 significantly inhibited both the phosphorylation of CREB and expression of TGFß2. In addition, treating Z12 or cancer cell lines with either of these 2 inhibitors significantly decreased their secretion of TGFß2. These data suggest that activated PI3 kinase and p38 MAPK play important roles in high expression of TGFß2 in cancer cells by stimulating the phosphorylation of CREB, which activates the CRE in the promoter of the TGFß2 gene. We have identified an important link between PI3 kinase, p38 MAPK, and TGFß2, providing an additional rationale for using inhibitors of these kinases as therapeutic drugs in cancer.


Assuntos
AMP Cíclico/metabolismo , Neoplasias/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Masculino , Morfolinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Elementos de Resposta/genética , Fator de Crescimento Transformador beta2/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
7.
Cardiovasc Res ; 87(4): 704-12, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20410290

RESUMO

AIMS: We have demonstrated an important role of bone marrow-derived stem cells in preservation of myocardial function. We investigated whether Akt-1 of lin(-)c-kit(+) stem cells preserves ventricular function following myocardial infarction (MI). METHODS AND RESULTS: Isolated lin(-)c-kit(+) cells were conjugated with anti-c-kit heteroconjugated to anti-vascular cell adhesion molecule to facilitate the attachment of stem cells into damaged tissues. Female severe combined immunodeficient mice were used as recipients. MI was created by ligation of the left descending artery. After 48 h, animals were divided into four groups: (i) sham (n = 5): animals underwent thoracotomy without MI; (ii) MI (n = 5): animals underwent MI and received medium; (iii) MI + wild-type (Wt) stem cells (n = 6): MI animals received 5 x 10(5) Wt lin(-)c-kit(+) stem cells; (iv) MI + Akt-1(-/-) stem cells (n = 6): MI animals received 5 x 10(5) Akt-1(-/-) lin(-)c-kit(+) stem cells. Two weeks later, left ventricular function was measured in the Langendorff mode. The peripheral administration of Wt armed stem cells into MI animals restored ventricular function, which was absent in animals receiving Akt-1(-/-) cells. Real-time PCR indicates a decrease in SRY3, a Y chromosome marker in hearts receiving Akt-1(-/-) cells. An increase in angiogenic response was demonstrated in hearts receiving Wt stem cells but not Akt-1(-/-) stem cells. CONCLUSION: Our results demonstrate that the peripheral administration of Wt lin(-)c-kit(+) stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1(-/-) stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.


Assuntos
Linhagem da Célula , Infarto do Miocárdio/cirurgia , Miocárdio/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transplante de Células-Tronco , Células-Tronco/enzimologia , Função Ventricular Esquerda , Animais , Anticorpos Biespecíficos , Cardiomegalia/enzimologia , Cardiomegalia/etiologia , Cardiomegalia/prevenção & controle , Adesão Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Recuperação de Função Fisiológica , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/imunologia
8.
Proc Natl Acad Sci U S A ; 107(1): 46-51, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20080798

RESUMO

NF-kappaB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-kappaB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptor-binding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-kappaB activity, and a high level of NSD1 activates NF-kappaB and reverses the inhibitory effect of FBXL11, whereas reduced expression of NSD1 decreases NF-kappaB activation. The targets are K218 and K221 of p65, which are methylated in cells with activated NF-kappaB. Overexpression of FBXL11 slowed the growth of HT29 cancer cells, whereas shRNA-mediated knockdown had the opposite effect, and these phenotypes were dependent on K218/K221 methylation. In mouse embryo fibroblasts, the activation of most p65-dependent genes relied on K218/K221 methylation. Importantly, expression of the FBXL11 gene is driven by NF-kappaB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-kappaB is an important element in the complex regulation of this key transcription factor.


Assuntos
Proteínas F-Box/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição RelA/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Embrião de Mamíferos/citologia , Proteínas F-Box/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oxirredutases N-Desmetilantes , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fator de Transcrição RelA/genética
9.
Proc Natl Acad Sci U S A ; 106(38): 16339-44, 2009 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-19805303

RESUMO

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10(-6) or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFkappaB. High levels of FBXL11 block the ability of NFkappaB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFkappaB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFkappaB-dependent signaling.


Assuntos
Proteínas F-Box/metabolismo , Mutagênese Insercional/métodos , NF-kappa B/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Northern Blotting , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas F-Box/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HT29 , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Lisina/metabolismo , Metilação , Microscopia de Fluorescência , Mutação , NF-kappa B/genética , Oxirredutases N-Desmetilantes/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
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