RESUMO
Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Citocinas/antagonistas & inibidores , Hepcidinas/metabolismo , Proteínas Musculares/antagonistas & inibidores , Talassemia/tratamento farmacológico , Animais , Anticorpos Neutralizantes/farmacologia , Linhagem Celular , Citocinas/química , Citocinas/metabolismo , Células HEK293 , Humanos , Ferro/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Domínios Proteicos/efeitos dos fármacos , Talassemia/metabolismoRESUMO
Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in ß-thalassemia. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, and ERFE suppresses hepatic hepcidin production through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for hepcidin control, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron. In vitro, ERFE decreased SMAD1, SMAD5, and SMAD8 phosphorylation and inhibited expression of BMP target genes. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6, and BMP7 but had little or no effect on hepcidin induction by BMP2, BMP4, BMP9, or activin B. A neutralizing anti-ERFE antibody prevented ERFE from inhibiting hepcidin induction by BMP5, BMP6, and BMP7. Cell-free homogeneous time-resolved fluorescence assays showed that BMP5, BMP6, and BMP7 competed with anti-ERFE for binding to ERFE. We conclude that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially impairing an evolutionarily closely related BMP subgroup of BMP5, BMP6, and BMP7. ERFE can act as a natural ligand trap generated by stimulated erythropoiesis to regulate the availability of iron.
Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Citocinas/metabolismo , Hepcidinas/metabolismo , Proteínas Musculares/metabolismo , Animais , Linhagem Celular , Células Hep G2 , Humanos , Ferro/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
Assuntos
Produtos Biológicos/química , Quimiocina CXCL13/antagonistas & inibidores , Modelos Moleculares , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos , Produtos Biológicos/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Humanos , Cinética , Mutação , Agregados Proteicos , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Solubilidade , Difração de Raios XRESUMO
Platelet derived growth factor-BB (PDGF-BB) is an important mitogen and cell survival factor during development. PDGF-BB binds PDGF receptor-ß (PDGFRß) to trigger receptor dimerization and tyrosine kinase activation. We present the pharmacological and biophysical characterization of a blocking PDGF-BB monoclonal antibody, MOR8457, and contrast this to PDGFRß. MOR8457 binds to PDGF-BB with high affinity and selectivity, and prevents PDGF-BB induced cell proliferation competitively and with high potency. The structural characterization of the MOR8457-PDGF-BB complex indicates that MOR8457 binds with a 2:1 stoichiometry, but that binding of a single MOR8457 moiety is sufficient to prevent binding to PDGFRß. Comparison of the MOR8457-PDGF-BB structure with that of the PDGFRß-PDGF-BB complex suggested the potential reason for this was a substantial bending and twisting of PDGF-BB in the MOR8457 structure, relative to the structures of PDGF-BB alone, bound to a PDGF-BB aptamer or PDGFRß, which makes it nonpermissive for PDGFRß binding. These biochemical and structural data offer insights into the permissive structure of PDGF-BB needed for agonism as well as strategies for developing specific PDGF ligand antagonists.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Proteínas Proto-Oncogênicas c-sis/antagonistas & inibidores , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/farmacologia , Aptâmeros de Peptídeos/química , Aptâmeros de Peptídeos/genética , Aptâmeros de Peptídeos/metabolismo , Aptâmeros de Peptídeos/farmacologia , Becaplermina , Sítios de Ligação de Anticorpos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas Proto-Oncogênicas c-sis/química , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de PlaquetasRESUMO
Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos/imunologia , Epitopos/imunologia , Proteínas tau/imunologia , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Encéfalo/metabolismo , Galinhas , Epitopos/química , Epitopos/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Dinoprostona/antagonistas & inibidores , Receptores Nucleares Órfãos/agonistas , Osteoartrite/complicações , Dor/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteína ADAMTS4 , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Ligantes , Receptores X do Fígado , Camundongos , Camundongos Mutantes , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/fisiologia , Osteoartrite/metabolismo , Dor/etiologia , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Prostaglandina-E Sintases , RatosRESUMO
The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody's activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.
Assuntos
Anticorpos Biespecíficos , Receptores de Enterotoxina , Linfócitos T , Humanos , Epitopos , Reconhecimento PsicológicoRESUMO
BACKGROUND: Nonsteroidal agonists have been developed that selectively bind to and activate estrogen receptor beta (ERbeta) rather than estrogen receptor alpha (ERalpha). ERbeta is expressed equally in both male and female mammals in multiple extragonadal tissues. Work reported elsewhere has demonstrated that ERbeta agonists have beneficial effects in multiple (but not all) models of inflammatory diseases and also increase survival in experimentally induced sepsis. METHODS: In these experiments, ERbeta agonists (ERB-041 or WAY-202196) were compared with vehicle control in the murine cecal ligation and puncture (CLP) model and in the pneumococcal pneumonia model of sepsis. The effect of WAY-202196 on the gene expression profile in the CLP model was further studied by transcriptome analysis of lung and small intestine tissue samples. RESULTS: ERbeta agonists provided a significant survival benefit in both experimental models of bacterial sepsis. This survival advantage was accompanied by reduced histologic evidence of tissue damage, reduced transcription of multiple proinflammatory proteins by transcriptome analysis and was not associated with increased bacterial outgrowth. CONCLUSIONS: ERbeta agonist administration provided a survival advantage in septic animals and appears to be a promising therapeutic modality in sepsis.
Assuntos
Receptor beta de Estrogênio/agonistas , Naftóis/uso terapêutico , Oxazóis/uso terapêutico , Sepse/tratamento farmacológico , Animais , Modelos Animais de Doenças , Receptor beta de Estrogênio/biossíntese , Receptor beta de Estrogênio/fisiologia , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Pneumocócica/tratamento farmacológico , Sepse/fisiopatologia , Transcrição Gênica/efeitos dos fármacosRESUMO
GDF15 is a distant TGF-ß family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Redução de Peso/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Glicosilação , Humanos , Camundongos , Mutação Puntual , Engenharia de ProteínasRESUMO
We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.
Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.
Assuntos
Agrecanas/química , Agrecanas/metabolismo , Serina Endopeptidases/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Agrecanas/análise , Agrecanas/imunologia , Alginatos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cartilagem/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Progressão da Doença , Epitopos/química , Epitopos/imunologia , Feminino , Regulação da Expressão Gênica , Ácido Glucurônico , Ácidos Hexurônicos , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Osteoartrite/metabolismo , Osteoartrite/patologia , Serina Endopeptidases/genéticaRESUMO
Peroxisome proliferator-activated receptor (PPAR)-gamma modulators, a class of antidiabetic drugs, have been associated with cardiovascular risks in type 2 diabetes in humans. The objective of this study was to explore possible cardiovascular risk biomarkers associated with PPAR-gamma in rodents that could provide an alert for risk to humans. Normal, myocardial infarction-induced heart failure (HF) or Zucker diabetic fatty (ZDF) rats were used. Rats (n = 5-6) were treated with either vehicle or rosiglitazone (RGZ; 3 or 45 mg/kg/day p.o.) for 4 weeks. Biomarkers for potential cardiovascular risks were assessed, including 1) ultrasound for cardiac structure and function; 2) neuroendocrine and hormonal plasma biomarkers of cardiovascular risk; 3) pharmacogenomic profiling of cardiac and renal tissue by targeted tissue low-density gene array representing ion channels and transporters, and components of the renin-angiotensin-aldosterone system; and 4) immunohistochemistry for cardiac fibrosis, hypertrophy, and inflammation (macrophages and tumor necrosis factor-alpha). HF was confirmed by increase in cardiac brain natriuretic peptide expression (p < 0.01) and echocardiography. Adequate exposure of RGZ was confirmed by pharmacokinetics (plasma drug levels) and the pharmacodynamic biomarker adiponectin. In normal or HF rats, RGZ had no negative effects on any of the biomarkers investigated. Similarly, RGZ had no significant effects on gene expression except for the increase in interleukin-6 mRNA expression in the heart and decrease in epithelial sodium channel beta in the kidney. In contrast, echocardiography showed improved cardiac structure and function after RGZ in ZDF rats. Taken together, this study suggests a limited predictive power of these preclinical models in respect to observed clinical adverse effects associated with RGZ.
Assuntos
Biomarcadores/sangue , Insuficiência Cardíaca/induzido quimicamente , Hipoglicemiantes/efeitos adversos , PPAR gama/agonistas , Tiazolidinedionas/efeitos adversos , Animais , Peso Corporal , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Ecocardiografia , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/patologia , Hemodinâmica/fisiologia , Hipoglicemiantes/farmacocinética , Imuno-Histoquímica , Miocardite/induzido quimicamente , Miocardite/patologia , Miocárdio/patologia , Tamanho do Órgão , RNA/genética , Ratos , Ratos Endogâmicos Lew , Ratos Zucker , Rosiglitazona , Tiazolidinedionas/farmacocinética , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND: Selective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under in vitro cross-linking and in vivo conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken. METHODS: In vitro antibody cross-linking and cell culture protocols reported for studies with a human agonistic antibody, TGN1412, were followed for Ab-01. rhIL21, the agonist ligand of the targeted receptor, and cross-linked anti-CD28 were used as positive controls for signal transduction. In vivo agonistic potential of Ab-01 was assessed by measuring expression levels of cytokine storm-associated and IL21 pathway genes in blood of cynomolgus monkeys before and after IV administration of Ab-01. RESULTS: Using a comprehensive set of assays that detected multiple activation signals in the presence of the positive control agonists, in vitro Ab-01-dependent activation was not detected in either PBMCs or the rhIL21-responsive cell line Daudi. Furthermore, no difference in gene expression levels was detected in blood before and after in vivo Ab-01 dosing of cynomolgus monkeys. CONCLUSIONS: Despite efforts to intentionally force an agonistic signal from Ab-01, none could be detected.
RESUMO
BACKGROUND: In preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study. METHODS: Genes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey. RESULTS: Stimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration. CONCLUSIONS: A robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.
Assuntos
Anticorpos/farmacologia , Bioensaio/métodos , Receptores de Interleucina-21/antagonistas & inibidores , Receptores de Interleucina-21/sangue , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Biomarcadores/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucinas/imunologia , Macaca fascicularis/imunologia , Receptores de Interleucina-21/imunologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TitulometriaRESUMO
PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Complexo CD3/imunologia , Neoplasias Colorretais/terapia , Neoplasias Gastrointestinais/terapia , Imunoterapia/métodos , Receptores de Enterotoxina/imunologia , Linfócitos T/imunologia , Transferência Adotiva/métodos , Animais , Anticorpos Biespecíficos/farmacocinética , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Feminino , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição TecidualRESUMO
Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.
Assuntos
Aterosclerose/tratamento farmacológico , LDL-Colesterol/efeitos dos fármacos , LDL-Colesterol/metabolismo , Indazóis/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Macaca fascicularis/metabolismo , Receptores Nucleares Órfãos/agonistas , Animais , Aterosclerose/metabolismo , Células CACO-2 , Cricetinae , Modelos Animais de Doenças , Humanos , Indazóis/sangue , Indazóis/química , Ligantes , Fígado/enzimologia , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos/metabolismoRESUMO
UNLABELLED: Methodological problems, including binding of myostatin to plasma proteins and cross-reactivity of assay reagents with other proteins, have confounded myostatin measurements. Here we describe development of an accurate assay for measuring myostatin concentrations in humans. Monoclonal antibodies that bind to distinct regions of myostatin served as capture and detector antibodies in a sandwich ELISA that used acid treatment to dissociate myostatin from binding proteins. Serum from myostatin-deficient Belgian Blue cattle was used as matrix and recombinant human myostatin as standard. The quantitative range was 0.15-37.50 ng/mL. Intra- and inter-assay CVs in low, mid, and high range were 4.1%, 4.7%, and 7.2%, and 3.9%, 1.6%, and 5.2%, respectively. Myostatin protein was undetectable in sera of Belgian Blue cattle and myostatin knockout mice. Recovery in spiked sera approximated 100%. ActRIIB-Fc or anti-myostatin antibody MYO-029 had no effect on myostatin measurements when assayed at pH 2.5. Myostatin levels were higher in young than older men (mean+/-S.E.M. 8.0+/-0.3 ng/mL vs. 7.0+/-0.4 ng/mL, P=0.03). In men treated with graded doses of testosterone, myostatin levels were significantly higher on day 56 than baseline in both young and older men; changes in myostatin levels were significantly correlated with changes in total and free testosterone in young men. Myostatin levels were not significantly associated with lean body mass in either young or older men. CONCLUSION: Myostatin ELISA has the characteristics of a valid assay: nearly 100% recovery, excellent precision, accuracy, and sufficient sensitivity to enable measurement of myostatin concentrations in men and women.
Assuntos
Androgênios/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Miostatina/sangue , Testosterona/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Androgênios/administração & dosagem , Animais , Bovinos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testosterona/administração & dosagem , Adulto JovemRESUMO
OBJECTIVE: Myostatin is an endogenous negative regulator of muscle growth and a novel target for muscle diseases. We conducted a safety trial of a neutralizing antibody to myostatin, MYO-029, in adult muscular dystrophies (Becker muscular dystrophy, facioscapulohumeral dystrophy, and limb-girdle muscular dystrophy). METHODS: This double-blind, placebo-controlled, multinational, randomized study included 116 subjects divided into sequential dose-escalation cohorts, each receiving MYO-029 or placebo (Cohort 1 at 1 mg/kg; Cohort 2 at 3 mg/kg; Cohort 3 at 10 mg/kg; Cohort 4 at 30 mg/kg). Safety and adverse events were assessed by reported signs and symptoms, as well as by physical examinations, laboratory results, echocardiograms, electrocardiograms, and in subjects with facioscapulohumeral dystrophy, funduscopic and audiometry examinations. Biological activity of MYO-029 was assessed through manual muscle testing, quantitative muscle testing, timed function tests, subject-reported outcomes, magnetic resonance imaging studies, dual-energy radiographic absorptiometry studies, and muscle biopsy. RESULTS: MYO-029 had good safety and tolerability with the exception of cutaneous hypersensitivity at the 10 and 30 mg/kg doses. There were no improvements noted in exploratory end points of muscle strength or function, but the study was not powered to look for efficacy. Importantly, bioactivity of MYO-029 was supported by a trend in a limited number of subjects toward increased muscle size using dual-energy radiographic absorptiometry and muscle histology. INTERPRETATION: This trial supports the hypothesis that systemic administration of myostatin inhibitors provides an adequate safety margin for clinical studies. Further evaluation of more potent myostatin inhibitors for stimulating muscle growth in muscular dystrophy should be considered.
Assuntos
Anticorpos/uso terapêutico , Toxidermias/epidemiologia , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/epidemiologia , Medição de Risco/métodos , Adulto , Estudos de Coortes , Comorbidade , Método Duplo-Cego , Feminino , Humanos , Incidência , Internacionalidade , Masculino , Efeito Placebo , Fatores de Risco , Resultado do TratamentoRESUMO
Patients exhibit a range of responses to drug treatment owing to individual genetic variation and biology. A deeper understanding of the human genome, enabled by increasingly powerful technologies to measure both its genes and gene products, has unleashed the concept of tailoring therapy to the individual patient upon pharmaceutical and clinical sciences. The successful application of personalized medicine depends upon the discovery and development of biomarkers. Biomarkers that either indicate pharmacodynamic effects or constitute predictive measures of individual patient responses can support dose selection and/or help determine therapeutic options. The development of biomarkers for clinical testing and validation can be facilitated by the use of ex vivo systems utilizing clinically relevant human tissues for the discovery of biomarkers of drug activity before first in human (FIH) studies. In this review we discuss the uses of ex vivo systems for both disease tissues and surrogate normal tissues to provide mechanistic insights into drug action and for the purpose of identifying candidate biomarkers.
Assuntos
Biomarcadores/análise , Tratamento Farmacológico , Animais , Células Sanguíneas/fisiologia , Cartilagem/metabolismo , Cartilagem/patologia , Células Cultivadas , Condrócitos/metabolismo , Resistência a Medicamentos , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Pele/citologia , Pele/efeitos dos fármacosRESUMO
Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.