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1.
Immunity ; 40(4): 477-89, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24745332

RESUMO

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.


Assuntos
Benzenoacetamidas/farmacologia , Compostos Benzidrílicos/farmacologia , Digoxina/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Redes Reguladoras de Genes/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Esclerose Múltipla/tratamento farmacológico , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Androstenóis/química , Animais , Benzenoacetamidas/química , Compostos Benzidrílicos/química , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Citocinas/metabolismo , Digoxina/química , Encefalomielite Autoimune Experimental/imunologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Biologia de Sistemas , Subpopulações de Linfócitos T/imunologia , Células Th17/imunologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
2.
J Immunol ; 192(6): 2564-75, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24516202

RESUMO

IL-17-producing CD4(+)Th17 cells, CD8(+)Tc17 cells, and γδ T cells play critical roles in the pathogenesis of autoimmune psoriasis. RORγt is required for the differentiation of Th17 cells and expression of IL-17. In this article, we describe a novel, potent, and selective RORγt inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time to our knowledge, provides a unique and powerful set of tools to probe RORγt-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated IL-17A production and inflammatory Th17-signature gene expression (Il17a, Il17f, Il22, Il26, Ccr6, and Il23) in mature human Th17 effector/memory T cells. In addition, TMP778, but not TMP776, inhibited IL-17A production in both human and mouse γδ T cells. IL-23-induced IL-17A production was also blocked by TMP778 treatment. In vivo targeting of RORγt in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, to our knowledge, we are the first to demonstrate that RORγt inverse agonists: 1) inhibit Tc17 cell differentiation, as well as IL-17 production by γδ T cells and CD8(+) Tc17 cells; 2) block imiquimod-induced cutaneous inflammation; 3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and 4) block IL-23-induced IL-17A expression. Thus, RORγt is a tractable drug target for the treatment of cutaneous inflammatory disorders, which may afford additional therapeutic benefit over existing modalities that target only IL-17A.


Assuntos
Dermatite/prevenção & controle , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células Th17/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Adulto , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Dermatite/imunologia , Dermatite/metabolismo , Relação Dose-Resposta a Droga , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Psoríase/sangue , Psoríase/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/imunologia , Pele/metabolismo , Pele/patologia , Bibliotecas de Moléculas Pequenas/química , Células Th17/imunologia , Células Th17/metabolismo , Transcriptoma/imunologia
3.
Immunology ; 145(3): 347-56, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25604624

RESUMO

The orphan nuclear receptor, retinoic acid receptor-related orphan nuclear receptor γt (RORγt), is required for the development and pathogenic function of interleukin-17A-secreting CD4(+) T helper type 17 (Th17) cells. Whereas small molecule RORγt antagonists impair Th17 cell development and attenuate autoimmune inflammation in vivo, the broader effects of these inhibitors on RORγt-dependent gene expression in vivo has yet to be characterized. We show that the RORγt inverse agonist TMP778 acts potently and selectively to block mouse Th17 cell differentiation in vitro and to impair Th17 cell development in vivo upon immunization with the myelin antigen MOG35-55 plus complete Freund's adjuvant. Importantly, we show that TMP778 acts in vivo to repress the expression of more than 150 genes, most of which fall outside the canonical Th17 transcriptional signature and are linked to a variety of inflammatory pathologies in humans. Interestingly, more than 30 genes are related with SMAD3, a transcription factor involved in the Th17 cell differentiation. These results reveal novel disease-associated genes regulated by RORγt during inflammation in vivo, and provide an early read on potential disease indications and safety concerns associated with pharmacological targeting of RORγt.


Assuntos
Diferenciação Celular/imunologia , Expressão Gênica/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Feminino , Adjuvante de Freund/imunologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imunização/métodos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/imunologia , Células Th17/efeitos dos fármacos , Células Th17/metabolismo
4.
Cell Chem Biol ; 30(3): 235-247.e12, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36863346

RESUMO

Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.


Assuntos
Neoplasias , Fatores de Transcrição , Animais , Humanos , Camundongos , Fator de Transcrição Ikaros , Imunoterapia , Neoplasias/terapia , Neoplasias/metabolismo , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição/metabolismo
5.
Immunother Adv ; 2(1): ltac019, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36196369

RESUMO

Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab. Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed. Results: Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9. Conclusion: Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms.

6.
J Immunother Cancer ; 10(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35217575

RESUMO

BACKGROUND: Lymphocyte-activation gene 3 (LAG-3) is an inhibitory immunoreceptor that negatively regulates T-cell activation. This paper presents preclinical characterization of the LAG-3 inhibitor, ieramilimab (LAG525), and phase I data for the treatment of patients with advanced/metastatic solid tumors with ieramilimab ±the anti-programmed cell death-1 antibody, spartalizumab. METHODS: Eligible patients had advanced/metastatic solid tumors and progressed after, or were unsuitable for, standard-of-care therapy, including checkpoint inhibitors in some cases. Patients received ieramilimab ±spartalizumab across various dose-escalation schedules. The primary objective was to assess the maximum tolerated dose (MTD) or recommended phase II dose (RP2D). RESULTS: In total, 255 patients were allocated to single-agent ieramilimab (n=134) and combination (n=121) treatment arms. The majority (98%) had received prior antineoplastic therapy (median, 3). Four patients experienced dose-limiting toxicities in each treatment arm across various dosing cohorts. No MTD was reached. The RP2D on a 3-week schedule was declared as 400 mg ieramilimab plus 300 mg spartalizumab and, on a 4-week schedule (once every 4 weeks; Q4W), as 800 mg ieramilimab plus 400 mg spartalizumab; tumor target (LAG-3) suppression with 600 mg ieramilimab Q4W was predicted to be similar to the Q4W, RP2D schedule. Treatment-related adverse events (TRAEs) occurred in 75 (56%) and 84 (69%) patients in the single-agent and combination arms, respectively. Most common TRAEs were fatigue, gastrointestinal, and skin disorders, and were of mild severity; seven patients experienced at least one treatment-related serious adverse event in the single-agent (5%) and combination group (5.8%). Antitumor activity was observed in the combination arm, with 3 (2%) complete responses and 10 (8%) partial responses in a mixed population of tumor types. In the combination arm, eight patients (6.6%) experienced stable disease for 6 months or longer versus six patients (4.5%) in the single-agent arm. Responding patients trended towards having higher levels of immune gene expression, including CD8 and LAG3, in tumor tissue at baseline. CONCLUSIONS: Ieramilimab was well tolerated as monotherapy and in combination with spartalizumab. The toxicity profile of ieramilimab in combination with spartalizumab was comparable to that of spartalizumab alone. Modest antitumor activity was seen with combination treatment. TRIAL REGISTRATION NUMBER: NCT02460224.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/farmacologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Pessoa de Meia-Idade , Adulto Jovem
8.
Cell Immunol ; 262(2): 150-61, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20206921

RESUMO

Cyclophosphamide in combination with immunotherapeutic approaches preferentially impinges on T(reg) activity and allows for robust generation of T cell effectors. Reduced dosages of cyclophosphamide are necessary to restrict its cytotoxic effects to the negative regulatory cell populations while sparing effector lymphocytes. We investigated cyclophosphamide dosing in combination with ZYC300, a PLG-encapsulated plasmid DNA vaccine which encodes the cytochrome P450 family member, CYP1B1, a known human tumor-associated antigen. In mice, three consecutive, low doses of cyclophosphamide comprised a superior regimen in enhancing the magnitude, diversity of epitopes, and avidity to individual epitopes of specific T cell responses when compared to regimens that used either a single low or a single high dose. Consecutive low doses of cyclophosphamide predominantly targeted T(regs) while sparing overall T lymphocyte counts. Thus, we report the synergistic activity of pharmacologic T(reg) depletion with cyclophosphamide on quantitatively and qualitatively increasing T cell responses to a known human tumor-associated antigen.


Assuntos
Ciclofosfamida/farmacologia , Imunossupressores/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Vacinas de DNA , Animais , Antígenos de Neoplasias/imunologia , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/imunologia , Linhagem Celular , Ciclofosfamida/imunologia , Citocromo P-450 CYP1B1 , Composição de Medicamentos , Epitopos/imunologia , Feminino , Humanos , Imunização , Imunossupressores/imunologia , Camundongos , Camundongos Endogâmicos C3H , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologia
9.
J Exp Med ; 211(1): 89-104, 2014 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24395888

RESUMO

IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença de Crohn/metabolismo , Regulação da Expressão Gênica/imunologia , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Doença de Crohn/imunologia , Citometria de Fluxo , Glucocorticoides/farmacologia , Humanos , Interferon gama/metabolismo , Análise em Microsséries , Células Th17/imunologia
10.
J Exp Med ; 208(9): 1875-87, 2011 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-21825017

RESUMO

Human memory T cells (T(M) cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6(+) and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common γ chain (γc)-using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6(+), but not CCR6(-), T(M) cells, even in CCR6(+) cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by γc-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6(+) T(M) cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment.


Assuntos
Citocinas/imunologia , Memória Imunológica/fisiologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores CCR6 , Transdução de Sinais/imunologia , Células Th17/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doença Crônica , Citocinas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/genética
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