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1.
Nat Commun ; 11(1): 4981, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020469

RESUMO

Antagonism or agonism of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) prevents weight gain and leads to dramatic weight loss in combination with glucagon-like peptide-1 receptor agonists in preclinical models. Based on the genetic evidence supporting GIPR antagonism, we previously developed a mouse anti-murine GIPR antibody (muGIPR-Ab) that protected diet-induced obese (DIO) mice against body weight gain and improved multiple metabolic parameters. This work reconciles the similar preclinical body weight effects of GIPR antagonists and agonists in vivo, and here we show that chronic GIPR agonism desensitizes GIPR activity in primary adipocytes, both differentiated in vitro and adipose tissue in vivo, and functions like a GIPR antagonist. Additionally, GIPR activity in adipocytes is partially responsible for muGIPR-Ab to prevent weight gain in DIO mice, demonstrating a role of adipocyte GIPR in the regulation of adiposity in vivo.


Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/uso terapêutico , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/patologia , Receptores dos Hormônios Gastrointestinais/deficiência , Receptores dos Hormônios Gastrointestinais/metabolismo
2.
Nat Biotechnol ; 38(9): 1073-1078, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32704169

RESUMO

A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination. The current gold standard is the conventional virus neutralization test requiring live pathogen and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization test that detects total immunodominant neutralizing antibodies targeting the viral spike (S) protein receptor-binding domain in an isotype- and species-independent manner. Our simple and rapid test is based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of patients with COVID-19 in two different countries, achieves 99.93% specificity and 95-100% sensitivity, and differentiates antibody responses to several human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it broadly accessible to the wider community for both research and clinical applications.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos/imunologia , Anticorpos/farmacologia , Betacoronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Testes de Neutralização , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/química
3.
Nat Commun ; 11(1): 1598, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221310

RESUMO

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.


Assuntos
Anticorpos/farmacologia , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Anticorpos/química , Membrana Celular/metabolismo , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Polímeros , Propilaminas , Ligação Proteica , Conformação Proteica
4.
Mol Carcinog ; 59(7): 691-700, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115801

RESUMO

Triple-negative breast cancer (TNBC) lacks a well-defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD-L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD-L1 protein's stability and immunosuppressive function. However, a strategy for PD-L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2-deoxy- d-glucose (2-DG), inhibits glycosylation of PD-L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib. Interestingly, 2-DG/gefitinib-induced deglycosylation of PD-L1 decreased the expression level of PD-L1 protein as well as its binding with PD-1. However, there was no significant decrease in 4-1BB expression and its binding with 4-1BBL by 2-DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2-DG/gefitinib and 4-1BB antibody enhances antitumor immunity in TNBC syngeneic murine models. Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD-L1 deglycosylation and 4-1BB stimulation in TNBC.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Desoxiglucose/farmacologia , Glucose/farmacologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Animais , Anticorpos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Gefitinibe/farmacologia , Células HEK293 , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C
5.
Diabetes ; 69(6): 1193-1205, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32198214

RESUMO

Obesity is a risk factor for type 2 diabetes (T2D); however, not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from donors with T2D and non-T2D (ND), especially obese donors (BMI ≥30 kg/m2). Islets from obese donors with T2D had reduced insulin secretion, decreased ß-cell exocytosis, and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis, and reduced granule docking. This was accompanied by reduced expression of the exocytotic proteins SNAP25, STXBP1, and VAMP2, likely because CD36 induced downregulation of the insulin receptor substrate (IRS) proteins, suppressed the insulin-signaling phosphatidylinositol 3-kinase/AKT pathway, and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human ß-cell line EndoC-ßH1 increased IRS1 and exocytotic protein levels, improved granule docking, and enhanced insulin secretion. Our results demonstrate that ß-cells from obese donors with T2D have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit ß-cell function in T2D associated with obesity.


Assuntos
Antígenos CD36/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Exocitose/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Obesidade/complicações , Anticorpos/farmacologia , Antígenos CD36/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/citologia
6.
Transfusion ; 60(4): 713-723, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32108957

RESUMO

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI. STUDY DESIGN AND METHODS: In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction. RESULTS: Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation. CONCLUSIONS: This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology.


Assuntos
Lesão Pulmonar Aguda Relacionada à Transfusão/prevenção & controle , Animais , Anticorpos/farmacologia , Plaquetas/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia
7.
Cancer Sci ; 111(5): 1840-1850, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32086991

RESUMO

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.


Assuntos
Anticorpos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Claudina-4/imunologia , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Claudina-1 , Claudina-4/química , Claudina-4/genética , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Lab Invest ; 100(2): 324-337, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896817

RESUMO

Abnormal Ca2+ handling is essential in the pathophysiology of degenerative muscle disorders, such as dilated cardiomyopathy (DCM) and muscular dystrophy (MD). Transient receptor potential cation channel, subfamily V, member 2 (TRPV2) is a candidate for Ca2+ entry and a potential therapeutic target for degenerative muscle disorders, there are few specific inhibitors for TRPV2. In this study, we produced a monoclonal antibody (designated mAb88-2) and two polyclonal antibodies (pAb591 and pAb592) that selectively recognize TRPV2 from the outside of cells and interact with the turret region of the pore-forming outer gate. These antibodies inhibited Ca2+ influx via TRPV2 in cultured cells and substantially reduced TRPV2 in the plasma membrane via cellular internalization. We evaluated the therapeutic efficacy of the functional antibody in δ-sarcoglycan-deficient hamster (J2N-k) models of DCM and MD and in the 4C30 DCM model of murine heart failure. The intraperitoneal administration of the functional antibody (0.5 mg/kg) for 2 weeks (once a week) prevented the progression of cardiac dysfunction, as evaluated by echocardiography and histological staining, and improved the abnormal Ca2+ handling (high diastolic Ca2+ level and small Ca2+ transient peak) in cardiomyocytes isolated from J2N-k hamsters and prevented skeletal muscle damage. Further, the antibody effectively prevented heart failure in the 4C30 mouse model with end-stage DCM. Interestingly, endogenous TRPV2 that accumulated in the cardiac and skeletal muscle sarcolemma disappeared upon antibody administration. Thus, the newly produced antibodies are capable of ameliorating DCM and MD by promoting the cellular internalization of TRPV2; antibodies specific to human TRPV2 may substantially improve the treatment of patients with degenerative muscle diseases.


Assuntos
Anticorpos , Canais de Cálcio , Cardiomiopatia Dilatada/metabolismo , Distrofias Musculares/metabolismo , Canais de Cátion TRPV , Animais , Anticorpos/química , Anticorpos/metabolismo , Anticorpos/farmacologia , Canais de Cálcio/metabolismo , Cricetinae , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
9.
Exp Eye Res ; 191: 107933, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935380

RESUMO

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 µg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α-smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.


Assuntos
Colágeno/metabolismo , Ceratócitos da Córnea/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Anticorpos/farmacologia , Células Cultivadas , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/efeitos dos fármacos , Substância Própria/citologia , Immunoblotting , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/imunologia , Fator de Crescimento Transformador beta/farmacologia
10.
Appl Microbiol Biotechnol ; 104(5): 1905-1914, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31940081

RESUMO

Glycosylation is a common post-translational modification that occurs during the production of antibodies. Glycans attached to antibodies play an important role in the pharmacokinetics, efficacy, and safety of therapeutic antibodies. In the modern antibody industry, it is important to adjust and control glycosylation modifications. The formation of specific sugar structures via glycosylation engineering is constantly evolving. This review summarizes the recent progress in glycosylation modifications, as well as the major discoveries and current understanding of the mechanisms involved, to provide new ideas for the research and development of therapeutic antibodies.


Assuntos
Anticorpos/metabolismo , Glicosilação , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/farmacologia , Humanos , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Controle de Qualidade
13.
BMC Infect Dis ; 19(1): 1031, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801478

RESUMO

BACKGROUND: Mycobacterium bovis (M. bovis) is the principal causative agent of bovine tuberculosis; however, it may also cause serious infection in human being. Type I IFN is a key factor in reducing viral multiplication and modulating host immune response against viral infection. However, the regulatory pathways of Type I IFN signaling during M. bovis infection are not yet fully explored. Here, we investigate the role of Type I IFN signaling in the pathogenesis of M. bovis infection in mice. METHODS: C57BL/6 mice were treated with IFNAR1-blocking antibody or Isotype control 24 h before M. bovis infection. After 21 and 84 days of infection, mice were sacrificed and the role of Type I IFN signaling in the pathogenesis of M. bovis was investigated. ELISA and qRT-PCR were performed to detect the expression of Type I IFNs and related genes. Lung lesions induced by M. bovis were assessed by histopathological examination. Viable bacterial count was determined by CFU assay. RESULTS: We observed an abundant expression of Type I IFNs in the serum and lung tissues of M. bovis infected mice. In vivo blockade of Type I IFN signaling reduced the recruitment of neutrophils to the lung tissue, mediated the activation of macrophages leading to an increased pro-inflammatory profile and regulated the inflammatory cytokine production. However, no impact was observed on T cell activation and recruitment in the early acute phase of infection. Additionally, blocking of type I IFN signaling reduced bacterial burden in the infected mice as compared to untreated infected mice. CONCLUSIONS: Altogether, our results reveal that Type I IFN mediates a balance between M. bovis-mediated inflammatory reaction and host defense mechanism. Thus, modulating Type I IFN signaling could be exploited as a therapeutic strategy against a large repertoire of inflammatory disorders including tuberculosis.


Assuntos
Interferon Tipo I/metabolismo , Mycobacterium bovis/patogenicidade , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo , Animais , Anticorpos/farmacologia , Citocinas/metabolismo , Feminino , Humanos , Interferon Tipo I/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Mycobacterium bovis/imunologia , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/efeitos dos fármacos
14.
Int J Nanomedicine ; 14: 9665-9675, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824158

RESUMO

Purpose: Vitamin D is a novel potential therapeutic agent for peritoneal dialysis (PD)-related peritoneal fibrosis, but it can induce hypercalcemia and vascular calcification, which limits its applicability. In this study, we create nanotechnology-based drug delivery systems to investigate its therapeutics and side effects. Materials and methods: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino-(polyethylene glycol)2000] (DSPE-PEG) and L-α-phosphatidylcholine (PC), which packages with 1α,25(OH)2D3, were used to construct vitamin D nanoliposomes. To confirm the function and safety of vitamin D nanoliposomes, peritoneal mesothelial cells were treated with TGF-ß1 and the reverse was attempted using vitamin D nanoliposomes. Antibodies (Ab) against the peritoneum-glycoprotein M6A (GPM6A) Ab were conjugated with vitamin D nanoliposomes. These particles were implanted into mice by intraperitoneal injection and the animals were monitored for the distribution and side effects induced by vitamin D. Results: Vitamin D nanoliposomes were taken up by the mesothelial cells over time without cell toxicity and it also provided the same therapeutic effect in vitro. In vivo study, fluorescent imaging showed vitamin D nanoliposomes allow specific peritoneum target effect and also ameliorate vitamin D side effect. Conclusion: Nanoliposomes vitamin D delivery systems for the prevention of PD-related peritoneal damage may be a potential clinical strategy in the future.


Assuntos
Nanomedicina , Diálise Peritoneal/efeitos adversos , Peritônio/patologia , Vitamina D/farmacologia , Animais , Anticorpos/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Liberação Controlada de Fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Cinética , Lipossomos , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Peritônio/efeitos dos fármacos , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Fator de Crescimento Transformador beta1/metabolismo
15.
Bioanalysis ; 11(24): 2283-2296, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31845602

RESUMO

Therapeutic proteins have the potential to induce unwanted immune responses. The potential impact of immunogenicity on pharmacokinetics, pharmacodynamics, safety and efficacy are well established. Here, we analyze key aspects of current US FDA and EMA guidelines on the development and validation of antidrug antibody assays. Although FDA and EMA guidance documents are in harmony on most points, EMA allows greater leeway for scientific judgement, while FDA recommends specific approaches that may not be appropriate in some situations. Many white papers suggest approaches different from the guidance documents, however, these can conflict with each other and are themselves only scientifically valid in certain situations. Here, we indicate when alternatives to guidance may be needed and what those approaches might be.


Assuntos
Anticorpos/uso terapêutico , Proteínas/uso terapêutico , United States Food and Drug Administration/normas , Anticorpos/farmacologia , Humanos , Proteínas/farmacologia , Estados Unidos
16.
Nat Commun ; 10(1): 5382, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772160

RESUMO

Accumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours.


Assuntos
Anticorpos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno HLA-A24/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteína Supressora de Tumor p53/genética , Animais , Anticorpos/genética , Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/efeitos adversos , Linhagem Celular Tumoral , Reações Cruzadas , Citotoxicidade Imunológica , Epitopos , Antígeno HLA-A24/genética , Antígeno HLA-A24/metabolismo , Humanos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Mutação , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 10(1): 5387, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772172

RESUMO

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.


Assuntos
Anticorpos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/metabolismo , Imunoterapia/métodos , Linfócitos T/imunologia , Animais , Anticorpos/genética , Antineoplásicos Imunológicos/imunologia , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Efeito Espectador , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Medicina de Precisão/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Immunol Res ; 67(4-5): 337-347, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31754971

RESUMO

The considerable variability of responses amongst subjects to disease triggers and immunotherapies is a major obstacle to designing better immune-based therapies. Therefore, development of patient-tailored precision medicine that improves the efficacy of immunomodulatory drugs is necessary. The individualized response to disease triggers and immunomodulatory therapies was studied using the concanavalin A (ConA) immune-mediated hepatitis model and the oral administration of anti CD3 or ß-glucosylceramide (GC). Mice were treated with anti-CD3 antibodies or GC followed by an injection of ConA. The effects of these treatments on liver damage and the immune profile were then analyzed. An individualized response to ConA and orally administered immunomodulatory agents was observed in eight consecutive experiments. While alleviation of the immune-mediated liver injury, as measured by serum levels of liver enzymes, was seen, and high intra-group and inter-experimental variabilities were detected. A similar individualized response was observed for the effect on serum levels of IFN-γ, TNF-α, and IL-10 and on CD4+CD25+, CD8+CD25+, and CD3+NK1.1+ lymphocytes. A personalized form of inherent randomness in an isolated system was documented, which may underlie the variability in responses to immune triggers and immunomodulatory therapies. The data support the use of personalized randomness-based platforms for improving the response to chronic therapies.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Fatores Imunológicos/farmacologia , Imunomodulação , Fígado/imunologia , Animais , Anticorpos/efeitos adversos , Anticorpos/farmacologia , Antígenos CD/imunologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Concanavalina A/efeitos adversos , Concanavalina A/farmacologia , Citocinas/imunologia , Fígado/patologia , Masculino , Camundongos
19.
Biomater Sci ; 8(1): 256-265, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31687671

RESUMO

Chemotherapy is a dominant treatment modality for different types and stages of cancer. However, hypoxia is one of the undesirable limitations of chemotherapy, which reduces the therapeutic efficiency in cancer treatment, ultimately leading to failure of the treatment. Herein, an ideal chemosensitization system capable of attenuating the tumor hypoxia microenvironment and enhancing chemotherapy effects in tumors was designed. This system (designated as the RA/RX Liposome) uses for the first time a pH-sensitive liposome to co-deliver cyclopeptide RA-V as chemotherapeutic drugs and antisense oligonucleotides as HIF-1α inhibitors (RX-0047) for attenuating tumor hypoxia, as well as a caspase-8 activation probe for therapeutic self-monitoring. After modification with death receptor 5-specific antibodies (anti-DR5) on the surface of the liposome, the RA/RX Liposome can successfully deliver components targeting colon tumors in vivo. This work should synergistically enhance the therapeutic effects of the treatment by successfully down-regulating HIF-1α expression against tumor hypoxia during the RA-V-induced apoptotic process. More importantly, the RA/RX Liposome can be precisely applied for therapeutic self-monitoring with the light-up fluorescence of the caspase-8 probe.


Assuntos
Anticorpos/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Oligonucleotídeos/administração & dosagem , Peptídeos Cíclicos/administração & dosagem , Hipóxia Tumoral/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Caspase 8/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Sinergismo Farmacológico , Feminino , Células HCT116 , Células HT29 , Humanos , Lipossomos , Camundongos , Oligonucleotídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
BMC Res Notes ; 12(1): 691, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653277

RESUMO

OBJECTIVE: Staphylococcus aureus and S. epidermidis as opportunistic pathogens, notable for their frequency and severity of infections are recognized as the most usual reasons for medical device-associated infections that strike hospitalized patients and also immunocompromised individuals. In this study, the polysaccharide intercellular adhesion (PIA) and Glycerol teichoic acid) Gly-TA) as two major macromolecules in the biofilm formation process were purified under the native condition and their structure was analyzed by using colorimetric assays and Fourier Transform Infrared spectroscopy (FTIR). Afterward, the immune response of macromolecules and the mixture of them were assessed by measuring total IgG titers. Subsequently, biofilm inhibitory effects of raising antibodies to biofilm former S. aureus and S. epidermidis were evaluated. RESULTS: Obtained data were shown a significant rise in levels of antibodies in immunized mice with mentioned antibodies in comparison with the control group. According to the obtained findings, mentioned antibodies could eliminate S. aureus and S. epidermidis biofilm formation in vitro assays. This survey confirms the proposal that immunization of mice with a mixture of Gly-TA and PIA vaccine could be secure and protected against S. epidermidis and S. aureus infection.


Assuntos
Anticorpos/imunologia , Aderência Bacteriana/imunologia , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/imunologia , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Ácidos Teicoicos/imunologia , Animais , Anticorpos/farmacologia , Biofilmes/efeitos dos fármacos , Colorimetria , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Espectroscopia de Infravermelho com Transformada de Fourier , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/fisiologia , Ácidos Teicoicos/metabolismo
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