RESUMO
Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.
Assuntos
Adenocarcinoma de Pulmão , Transportador de Glucose Tipo 1 , Neoplasias Pulmonares , Linfócitos do Interstício Tumoral , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Prognóstico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Masculino , Feminino , Idoso , Regulação Neoplásica da Expressão Gênica , Pessoa de Meia-Idade , Perfilação da Expressão GênicaRESUMO
INTRODUCTION: Type 1 and type 3 von Willebrand disease (VWD) are caused by partial and complete, quantitative deficiency of von Willebrand factor (VWF), respectively, and factor (F)VIII/VWF complex concentrates are used for haemostatic treatment. Emicizumab, mimics activated FVIII, reduces bleeding in haemophilia A patients. The effects of emicizumab on haemostasis in both types of VWD remain to be fully established, however. AIM: To examine the effects of emicizumab on thrombogenesis in type 1 and type 3 VWD. PATIENTS/METHODS: Perfusion chamber experiments under high shear conditions (2500 s-1 ) combined with immunostaining were performed using whole blood samples from patients with type 1 (VWF:Ag 25 U/dl) and type 3 VWD (<1.0 U/dl). RESULTS: The addition of FVIII (1 U/ml) to type 1 blood did not affect thrombus formation, whilst supplementation with VWF (1.6 U/ml) or FVIII/VWF (1 U/ml/1.6 U/ml) enhanced thrombogenesis to a similar extent. FVIII/VWF promoted thrombus formation significantly more than VWF alone, however, in type 3 blood. Emicizumab (100 µg/ml) augmented thrombus formation in type 3 blood compared to FVIII, and this potency seemed to be somewhat greater than that of VWF. Surface coverage of formed thrombus in type 3 VWD was less than that in type 1 VWD, but thrombus height was comparable in both. The addition of emicizumab to type 3 blood enhanced thrombin generation and fibrin formation compared to control IgG. CONCLUSION: Emicizumab promoted mechanisms of thrombus formation in vitro in type 3 and type 1 VWD, suggesting the possibility of alternative therapeutic protocols in these patients.
Assuntos
Trombose , Doença de von Willebrand Tipo 1 , Doença de von Willebrand Tipo 3 , Doenças de von Willebrand , Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Fator VIII/uso terapêutico , Humanos , Trombose/tratamento farmacológico , Doença de von Willebrand Tipo 3/tratamento farmacológico , Doenças de von Willebrand/tratamento farmacológico , Fator de von Willebrand/uso terapêuticoRESUMO
INTRODUCTION: Type 2A von Willebrand disease (VWD) is common in type-2 group caused by qualitative deficiency of von Willebrand factor (VWF). Emicizumab is a bispecific antibody that mimics activated factor VIII (FVIIIa) cofactor function, and emicizumab prophylaxis substantially reduces bleeding in patients with haemophilia A. It is unknown whether emicizumab affects thrombus formation in type 2A VWD characterized by not only low FVIII levels but also the impaired platelet adhesion and aggregation. AIM: To examine the coagulant potential of emicizumab in type 2A VWD. PATIENTS/METHODS: Perfusion chamber experiments combined with immunostaining were performed using whole blood from 5 patients with type 2A VWD under high shear condition (2500 s-1 ). RESULTS: The addition of FVIII to type 2A VWD whole blood did not augment thrombus formation, whilst supplementation with VWF or FVIII/VWF enhanced. FVIII appeared to contribute to thrombus height rather than surface coverage. The addition of emicizumab enhanced thrombus formation in type 2A VWD compared with FVIII, but this potency was less than the presence of VWF. The effect on thrombus formation mediated by emicizumab appeared to be more rapid than that by FVIII for non-requirement of activation step of FVIII, whilst that by FVIII showed more impact on thrombus formation at the late phase. CONCLUSION: Emicizumab-induced enhancing effects of thrombus formation, independent on VWF, may be useful as an alternative therapy for type 2A VWD patients. These results supported a critical role for the FVIII-VWF complex facilitating thrombus formation under high shear.
Assuntos
Anticorpos Biespecíficos , Trombose , Doença de von Willebrand Tipo 2 , Doenças de von Willebrand , Anticorpos Monoclonais Humanizados , Fator VIII , Humanos , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/prevenção & controle , Doença de von Willebrand Tipo 2/tratamento farmacológico , Fator de von WillebrandRESUMO
Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti-FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once-weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab-mediated haemostasis remain to be explored. We investigated the role of APC-mediated reactions in these circumstances. APC dose-dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII-deficient plasmas, and in normal plasmas preincubated with an anti-FVIII antibody (FVIII-depleted). FVIIIa-independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC-induced down-regulation of emicizumab-dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII-depleted FV-deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab-related TG in FVIII-depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab-driven haemostasis was down regulated by APC-mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Hemofilia A/sangue , Hemostasia/efeitos dos fármacos , Hemostáticos/farmacologia , Proteína C/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fator VIIIa/metabolismo , Fator Va/metabolismo , Humanos , Proteína C/administração & dosagem , Trombina/biossínteseRESUMO
ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator IXa/imunologia , Fator X/imunologia , Hemofilia A/complicações , Hemorragia/complicações , Hemorragia/prevenção & controle , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular , Modelos Animais de Doenças , Hemorragia/patologia , Humanos , Articulações/efeitos dos fármacos , Articulações/patologia , Macaca fascicularis , Masculino , CamundongosRESUMO
Background: Activated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. Objectives: To investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. Methods: In pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 µg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. Results: The APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. Conclusion: NXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.
RESUMO
BACKGROUND: The hemostatic effect of recombinant (r) factor (F)VIIa after repetitive intermittent administration may be attenuated in patients with hemophilia A (PwHA) with inhibitors (PwHAwI) creating a clinically unresponsive status, although mechanism(s) remain to be clarified. In patients receiving prophylaxis treatment with emicizumab, concomitant rFVIIa is sometimes utilized in multiple doses for surgical procedures or breakthrough bleeding. AIM AND METHODS: We identified 'unresponsiveness' to rFVIIa, based on global coagulation function monitored using rotational thromboelastometry (ROTEM) in 11 PwHAwI and 5 patients with acquired HA, and investigated possible mechanisms focusing on the association between plasma FX levels and rFVIIa-mediated interactions. RESULTS: Our data demonstrated that FX antigen levels were lower in the rFVIIa-unresponsive group than in the rFVIIa-responsive group (0.46 ± 0.14 IU/mL vs. 0.87 ± 0.15 IU/mL, p < 0.01). This relationship was further examined by thrombin generation assays using a FX-deficient PwHAwI plasma model. The addition of FX with rFVIIa was associated with increased peak thrombin (PeakTh) generation. At low levels of FX (<0.5 IU/mL), rFVIIa failed to increase PeakTh to the normal range, consistent with clinical rFVIIa-unresponsiveness. In the presence of emicizumab (50 µg/mL), PeakTh was increased maximally to 80 % of normal, even at low levels of FX (0.28 IU/mL). CONCLUSIONS: Unresponsiveness to rFVIIa was associated with reduced levels of FX in PwHAwI. Emicizumab exhibited in vitro coagulation potential in the presence of FX at concentrations that appeared to limit the clinical response to rFVIIa therapy.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Fator VIIa , Fator X , Hemofilia A , Hemostasia , Proteínas Recombinantes , Feminino , Humanos , Masculino , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator VIIa/farmacologia , Fator VIIa/uso terapêutico , Fator X/metabolismo , Hemofilia A/tratamento farmacológico , Hemofilia A/sangue , Hemostasia/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , TromboelastografiaRESUMO
Patients with hemophilia A (PwHA) may have concurrent deficiency of representative anticoagulant proteins, protein (P)C, PS, and antithrombin (AT), which reduces bleeding frequency. However, emicizumab-driven hemostasis in PwHA with such thrombophilic potential remains unclarified. This study investigated the influence of natural anticoagulants on emicizumab-driven coagulation in HA model plasma. Various concentrations of PS and AT were added to PS-deficient plasma and AT-deficient plasma in the presence of anti-FVIII antibody (FVIIIAb; 10BU/mL). PC-deficient plasma was mixed with normal plasma at various concentrations in the presence of FVIIIAb. Emicizumab (50 µg/mL) was added to these thrombophilic HA model plasmas, prior to tissue factor/ellagic acid-triggered thrombin generation assays. Co-presence of emicizumab increased peak thrombin values (PeakTh) dependent on PS, AT, and PC concentrations. Maximum coagulation potentials in the PS-reduced HA model plasmas remained normal in the presence of emicizumab. PeakTh were close to normal in the presence of 50%AT irrespective of emicizumab, but were higher than normal in the presence of 25%AT. Addition of recombinant FVIIa (corresponding to an administered dose of 90 µg/kg) enhanced coagulation potential to normal levels. Our findings provide novel information on hemostatic regulation in emicizumab-treated PwHA with a possible thrombophilic disposition.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Hemofilia A , Hemostáticos , Trombofilia , Humanos , Fator VIII , Trombina/metabolismo , Hemostasia , Hemofilia A/tratamento farmacológico , Anticoagulantes/uso terapêutico , Trombofilia/tratamento farmacológico , Antitrombinas/farmacologiaRESUMO
BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.
Assuntos
Anticorpos Biespecíficos , Hemofilia A , Hemostáticos , Humanos , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Trombina/metabolismo , Hemostasia , Coagulação Sanguínea , Fator VIIIRESUMO
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
Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.
Assuntos
Anticorpos Biespecíficos , Hemofilia A , Animais , Engenharia de Proteínas/métodos , Linhagem Celular , Dimerização , MamíferosRESUMO
In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.
Assuntos
Doença Celíaca , Glutens , Camundongos , Animais , Humanos , Coelhos , Glutens/química , Anticorpos Neutralizantes , Antígenos HLA-DQ , Peptídeos/química , Epitopos/química , Camundongos TransgênicosRESUMO
Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/metabolismo , Linfócitos do Interstício Tumoral , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 µg/kg). The lowest dose of 0.1 µg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927.
Assuntos
Anticorpos Biespecíficos , Glipicanas , Linfócitos T , Anticorpos Biespecíficos/administração & dosagem , Relação Dose-Resposta Imunológica , Glipicanas/imunologia , Humanos , Linfócitos T/imunologiaRESUMO
Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Neoplasias , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/farmacologia , Capecitabina , Humanos , Neoplasias/tratamento farmacológico , Linfócitos TRESUMO
BACKGROUND: Emicizumab is a bispecific antibody to factor (F) IXa and FX that mimics the FVIIIa cofactor function. Emicizumab prophylaxis markedly decreases bleeding episodes in patients with hemophilia A (PwHAs), irrespective of the presence of FVIII inhibitors. However, thrombotic microangiopathy (TMA) was reported when repeated high doses of activated prothrombin complex concentrates (aPCC) were concomitantly used with emicizumab. Although bypassing agents (BPAs) are vital in the hemostatic treatment for PwHAs with inhibitors, the mechanism of emicizumab-related TMA remains unclear. AIM: To assess the risk of excessive thrombus formation associated with BPAs and emicizumab under high shear conditions. METHODS: Perfusion flow-chamber experiments under high shear conditions were performed using whole blood from PwHAs in the presence of emicizumab without or together with FVIII or BPAs ex vivo. RESULTS: Emicizumab (100 µg/mL) added ex vivo to whole blood from PwHAs improved defective thrombus formation in a similar manner to that observed with the addition of recombinant FVIII at the early phase, while FVIII continued to be important at the later stages. aPCC (1.2 U/mL equivalent to 100 U/kg) or recombinant FVIIa (1.1 µg/mL; equivalent to 90 µg/kg) together with emicizumab further promoted platelet interactions and fibrin formation ex vivo but did not induce excessive thrombus formation. CONCLUSION: Emicizumab enhanced thrombin generation at local sites and improved defective hemostasis in whole blood from PwHAs under high shear conditions. Simple concomitant use of BPAs with emicizumab did not mediate excessive thrombus formation and remains an option for hemostatic management of emicizumab-treated PwHAs with inhibitors.
Assuntos
Anticorpos Biespecíficos/efeitos adversos , Anticorpos Monoclonais Humanizados/efeitos adversos , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/tratamento farmacológico , Trombose/induzido quimicamente , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Hemofilia A/sangue , Humanos , Tempo de Tromboplastina Parcial , Trombose/sangueRESUMO
INTRODUCTION: Type 2N von Willebrand disease (2NVWD) is characterized by a mild to moderate reduction in plasma levels of factor (F)VIII associated with defective binding of von Willebrand factor (VWF) to FVIII and accelerated proteolysis and clearance of FVIII. The clinical phenotype in 2NVWD is often indistinguishable from mild/moderate hemophilia (H)A. Emicizumab is a bispecific antibody to FIX/FIXa and FX/FXa that mimics FVIIIa cofactor function, and emicizumab prophylaxis significantly reduces bleeding events in patients with severe HA. AIM: We investigated the potential benefits of emicizumab in the hemostatic management of 2NVWD. PATIENTS/METHODS: Perfusion chamber experiments were performed using whole blood from three 2NVWD patients with different clinical phenotypes (bleeding scores: 0, 6 and 20; mutations: p.R816W, p.R816W, and p.R365X/p.T791M, respectively). Furthermore, the impact of specific FVIII-VWF interactions on thrombus formation was investigated. RESULTS: Defective thrombus formation that correlated with bleeding phenotype was evident in these 2NVWD patients. Emicizumab improved surface coverage and thrombus height in all cases. Multi-color immunostaining of thrombi further demonstrated that emicizumab enhanced thrombin generation and fibrin formation. The addition of FVIII alone to 2NVWD whole blood did not augment thrombus formation, while supplementation with FVIII/VWF complex enhanced platelet-fibrin interactions. Furthermore, an anti-FVIII monoclonal antibody known to interrupt the release of FVIIIa from VWF depressed these effects. CONCLUSIONS: Emicizumab-induced enhancing effects of thrombus formation, independent on VWF, might be useful as an alternative therapy for 2NVWD patients. The extent of FVIII-VWF interaction should be optimal to deliver and release FVIII/FVIIIa on the activated platelet surface.
Assuntos
Trombose , Doença de von Willebrand Tipo 2 , Doenças de von Willebrand , Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Fator VIII , Fator VIIIa , Humanos , Trombose/tratamento farmacológico , Fator de von WillebrandRESUMO
Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.
Assuntos
Anticorpos Monoclonais/farmacologia , Força Muscular/efeitos dos fármacos , Doenças Musculares/fisiopatologia , Miostatina/imunologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Diferenciação de Crescimento/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Tamanho do Órgão , Transdução de SinaisRESUMO
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Trifosfato de Adenosina , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.