RESUMO
Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called TmodTM, that contains an activating receptor (activator) and a separate inhibitory receptor (blocker). In proof-of-concept experiments, we use CD19 as the activating antigen and HLA-A*02 as the blocker antigen. This specific Tmod system, which employs a blocker targeting a ubiquitously expressed HLA class I antigen to inhibit CAR activation, has an inherent mechanism for selectivity/safety, designed to activate only when a specific HLA class I antigen is lost. Nonetheless, it is important to test off-target reactivity in functional assays, especially given the disconnect between ligand-binding and function among T cell receptors (TCRs) and chimeric antigen receptors (CARs). We show these cell-based assays yield consistent results with high sensitivity and specificity. The general strategy is likely applicable to more traditional single-receptor CAR- and TCR-T therapeutics.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/fisiologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Deleção de Genes , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Here, we show that a NOT gated cell therapy (Tmod) can exploit antigens such as epidermal growth factor receptor (EGFR) and human leukocyte antigen-E (HLA-E) which are widely expressed on cancer cells. Noncancerous cells-despite high expression of these antigens-are protected from cytotoxicity by the action of an inhibitory receptor ("blocker") via a mechanism that involves blocker modulation of CAR surface expression. The blocker is triggered by the product of a polymorphic HLA allele (e.g., HLA-A∗02) deleted in a significant subset of solid tumors via loss of heterozygosity. Moreover, Tmod constructs that target mouse homologs of EGFR or HLA-E for activation, and a mouse-equivalent of HLA-A∗02 for inhibition, protect mice from toxicity caused by the CAR alone. The blocker also controls graft vs. host response in allogeneic T cells in vitro, consistent with the use of Tmod cells for off-the-shelf therapy without additional gene-editing.
RESUMO
Structure-activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38α mitogen activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency was observed for 2,6-disubstituted phenyl moieties and N-ethyl triazolopyridazinone cores due to key contacts with Leu108, Ala157 and Val38. Further investigation led to the identification of three compounds, 3g, 3j and 3m that are highly potent inhibitors of LPS-induced MAPKAP kinase 2 (MK2) phosphorylation in 50% human whole blood (hWB), and possess desirable in vivo pharmacokinetic and kinase selectivity profiles.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Triazóis/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piridazinas/síntese química , Piridazinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.
Assuntos
Neoplasias Colorretais , Receptores de Antígenos Quiméricos , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Antígeno HLA-A2/genética , Humanos , Perda de HeterozigosidadeRESUMO
A novel class of pyrazolopyridazine p38alpha mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC(50) values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38alpha inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38alpha/beta over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of ca. 0.08mg/kg.
Assuntos
Anti-Inflamatórios/química , Benzamidas/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridazinas/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Benzamidas/síntese química , Benzamidas/farmacocinética , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Interleucina-8 , Lipopolissacarídeos/toxicidade , Masculino , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/síntese química , Pirazóis/farmacocinética , Piridazinas/síntese química , Piridazinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A novel class of fused pyrazole-derived inhibitors of p38alpha mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC(50) values in the low single-digit nanomolar range in whole blood. Further investigation of their pharmacokinetic profiles led to the identification of two potent and orally bioavailable p38 inhibitors 10 m and 10 q. Inhibitor 10 m was found to be efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of 0.1mg/kg while 10 q was found to have an ED(50) of 0.05-0.07 mg/kg.
Assuntos
Anti-Inflamatórios/química , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridonas/química , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Cristalografia por Raios X , Humanos , Interleucina-8/sangue , Lipopolissacarídeos/farmacologia , Masculino , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Piridonas/administração & dosagem , Piridonas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.
Assuntos
Antirreumáticos/síntese química , Piridazinas/síntese química , Piridonas/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antirreumáticos/farmacocinética , Antirreumáticos/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Sítios de Ligação , Colágeno , Feminino , Humanos , Interleucina-8/biossíntese , Interleucina-8/sangue , Lipopolissacarídeos/farmacologia , Masculino , Modelos Moleculares , Fosforilação , Piridazinas/farmacocinética , Piridazinas/farmacologia , Piridonas/farmacocinética , Piridonas/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The p38alpha mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50) < or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.
Assuntos
Descoberta de Drogas , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Piridonas/administração & dosagem , Piridonas/farmacologia , Administração Oral , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Colágeno/farmacologia , Humanos , Masculino , Proteína Quinase 14 Ativada por Mitógeno/química , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Piridonas/síntese química , Piridonas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-alpha inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.
Assuntos
Descoberta de Drogas , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/farmacologia , Talidomida/análogos & derivados , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Humanos , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Talidomida/administração & dosagem , Talidomida/química , Talidomida/farmacologiaRESUMO
The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.
Assuntos
Aminas/química , Benzeno/química , Isoxazóis/administração & dosagem , Isoxazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Ftalazinas/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/enzimologia , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Isoxazóis/química , Isoxazóis/uso terapêutico , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Relação Estrutura-AtividadeRESUMO
Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Ftalazinas/química , Ftalazinas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Células Cultivadas , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Ftalazinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacologia , Ratos , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.