RESUMO
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Assuntos
Complexo CD3 , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/terapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Complexo CD3/imunologia , Animais , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Ativação Linfocitária/imunologia , Feminino , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2.
Assuntos
Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Dados de Sequência Molecular , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Serina/genética , Serina/metabolismoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified in pedigrees of autosomal-dominant familial Parkinson's disease (PARK8). It has been shown that the kinase activity of LRRK2 is required for its neuronal toxicity, although how familial Parkinson mutations affect the function of LRRK2 has not been well characterized. In the present study, we systematically characterized the autophosphorylation of LRRK2 by phosphopeptide mapping and identified Thr1348, Thr1349, and Thr1357 as the major autophosphorylation sites. We found that the autophosphorylation at Thr1357 is downregulated by the Y1699C mutation, possibly through a conformational alteration of the ROC domain. We also found that I2020T mutant LRRK2 undergoes excessive autophosphorylation in cell lysates in vitro at a low concentration of ATP. These results highlight the differential effects of familial mutations in LRRK2 on its conformation and enzymatic properties.
Assuntos
Mutação , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Cromatografia em Camada Fina , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mapeamento de Peptídeos , Fosfopeptídeos/química , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Treonina/químicaRESUMO
Parkinson's disease (PD) is a major adult-onset neurodegenerative disorder affecting the extrapyramidal motor system. A subset of patients develop PD as an autosomal dominant trait, of which PARK8 caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is highlighted because of its high frequency and clinicopathological similarity to sporadic PD. Previous studies have suggested that overactivation of LRRK2 caused by missense mutations leads to neuronal toxicity in PARK8, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (DeltaN-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in Sf9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/ionization- time of flight (MALDI-TOF) mass spectrometer. We identified Ser1403, Thr1404, Thr1410, Thr1491 located within the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, as the autophosphorylation sites. Substitution of Thr1967, an autophosphorylation site located within the kinase domain, to Ala caused a significant decrease in the kinase activity, implicating Thr1967 in the kinase activity of LRRK2. Phosphospecific antibodies to the autophosphorylation sites specifically recognized full-length LRRK2 subjected to in vitro phosphorylation reaction, indicating that the autophosphorylation takes place in holoproteins. Further analysis of autophosphorylation will clarify the mechanism of activation of LRRK2, as well as the pathomechanism of PD in relation to overactivation of LRRK2.