RESUMEN
PURPOSE: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody. EXPERIMENTAL DESIGN: This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2). RESULTS: FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood. CONCLUSIONS: This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos CD/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Animales , Afinidad de Anticuerpos , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Ratones , Unión Proteica , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2, were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain. mAb2 containing anti-LAG-3 Fcabs have mAb-like biophysical characteristics and retain LAG-3 binding and functional activity. mAb2 can thus be generated using multiple Fabs to investigate bispecific parings and develop novel therapeutics.
Asunto(s)
Anticuerpos Biespecíficos , Antígenos CD/inmunología , Fragmentos Fc de Inmunoglobulinas , Animales , Humanos , Macaca fascicularis/metabolismo , Ratones , Ingeniería de Proteínas , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
After phagocytosis by macrophages, Staphylococcus aureus evades killing in an α-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like MCL-1 (myeloid cell leukemia-1). Here, using purified α-toxin and a set of hla-deficient strains, we show that α-toxin is critical for the induction of MCL-1 expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with α-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of α-toxin by intracellular bacteria. The increased expression of MCL-1 in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of S. aureus strains secreting α-toxin. Thus, S. aureus recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by α-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of S. aureus depends on α-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.
Asunto(s)
Toxinas Bacterianas/metabolismo , Citoprotección , Proteínas Hemolisinas/metabolismo , Macrófagos/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Toxinas Bacterianas/genética , Células Cultivadas , Proteínas Hemolisinas/genética , Humanos , Evasión Inmune , Interleucina-6/metabolismo , Macrófagos/microbiología , Mutación/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , FN-kappa B/metabolismo , Fagocitosis , Infecciones Estafilocócicas/transmisión , Staphylococcus aureus/patogenicidad , Receptor Toll-Like 2/metabolismo , Factores de VirulenciaRESUMEN
It has been reported that IL-6 knockout mice (IL-6â»/â») possess lower endurance capacity than wild type mice (WT), however the underlying mechanism is poorly understood. The aim of the present work was to examine whether reduced endurance running capacity in IL-6â»/â» mice is linked to impaired maximal oxygen uptake (V'O(2max)), decreased glucose tolerance, endothelial dysfunction or other mechanisms. Maximal running velocity during incremental running to exhaustion was significantly lower in IL-6â»/â» mice than in WT mice (13.00±0.97 m·min⻹ vs. 16.89±1.15 m·min⻹, P<0.02, respectively). Moreover, the time to exhaustion during running at 12 m·min⻹ in IL-6â»/â» mice was significantly shorter (P<0.05) than in WT mice. V'O(2max) in IL-6â»/â» (nâ=â20) amounting to 108.3±2.8 ml·kg⻹·min⻹ was similar as in WT mice (nâ=â22) amounting to 113.0±1.8 ml·kg⻹·min⻹, (Pâ=â0.16). No difference in maximal COX activity between the IL-6â»/â» and WT mice in m. soleus and m. gastrocnemius was found. Moreover, no impairment of peripheral endothelial function or glucose tolerance was found in IL-6â»/â» mice. Surprisingly, plasma lactate concentration during running at 8 m·min⻹ as well at maximal running velocity in IL-6â»/â» mice was significantly lower (P<0.01) than in WT mice. Interestingly, IL-6â»/â» mice displayed important adaptive mechanisms including significantly lower oxygen cost of running at a given speed accompanied by lower expression of sarcoplasmic reticulum Ca²âº-ATPase and lower plasma lactate concentrations during running at submaximal and maximal running velocities. In conclusion, impaired endurance running capacity in IL-6â»/â» mice could not be explained by reduced V'O(2max), endothelial dysfunction or impaired muscle oxidative capacity. Therefore, our results indicate that IL-6 cannot be regarded as a major regulator of exercise capacity but rather as a modulator of endurance performance. Furthermore, we identified important compensatory mechanism limiting reduced exercise performance in IL-6â»/â» mice.
Asunto(s)
Endotelio/fisiología , Interleucina-6/genética , Consumo de Oxígeno , Condicionamiento Físico Animal , Resistencia Física/genética , Animales , Temperatura Corporal , Citrato (si)-Sintasa/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Tolerancia al Ejercicio , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Canales Iónicos/metabolismo , Ácido Láctico/sangre , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteína Desacopladora 3RESUMEN
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.