RESUMO
Given the clinical success of cytokine blockade in managing diverse inflammatory human conditions, this approach could be exploited for numerous refractory or uncontrolled inflammatory conditions by identifying novel targets for functional blockade. Interleukin (IL)-18, a pro-inflammatory cytokine, is relatively underestimated as a therapeutic target, despite accumulated evidence indicating the unique roles of IL-18 in acute and chronic inflammatory conditions, such as macrophage activation syndrome. Herein, we designed a new form of IL-18 blockade, i.e., APB-R3, a long-acting recombinant human IL-18BP linked to human albumin-binding Fab fragment, SL335, for extending half-life. We then explored the pharmacokinetics and pharmacodynamics of APB-R3. In addition to an extended serum half-life, APB-R3 alleviates liver inflammation and splenomegaly in a model of the macrophage activation syndrome induced in IL-18BP knockout mice. Moreover, APB-R3 substantially controlled skin inflammation in a model of atopic dermatitis. Thus, we report APB-R3 as a new potent IL-18 blocking agent that could be applied to treat IL-18-mediated inflammatory diseases.
Assuntos
Dermatite Atópica , Síndrome de Ativação Macrofágica , Camundongos , Animais , Humanos , Dermatite Atópica/tratamento farmacológico , Interleucina-18/uso terapêutico , Albumina Sérica Humana/uso terapêutico , Síndrome de Ativação Macrofágica/tratamento farmacológico , Citocinas/uso terapêutico , Fatores Imunológicos/uso terapêutico , InflamaçãoRESUMO
Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.
Assuntos
Anticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/sangue , Proteínas Recombinantes/uso terapêutico , Albumina Sérica Humana/química , Albumina Sérica Humana/imunologia , Anticorpos/química , Reações Cruzadas , Meia-Vida , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Ligação Proteica , Receptores Fc/metabolismoRESUMO
Introduction: Administration of follicle-stimulating hormone (FSH) has been recommended to stimulate spermatogenesis in infertile men with hypogonadotropic hypogonadism, whose sperm counts do not respond to human chorionic gonadotropin alone. However, FSH has a short serum half-life requiring frequent administration to maintain its therapeutic efficacy. To improve its pharmacokinetic properties, we developed a unique albumin-binder technology, termed "anti-serum albumin Fab-associated" (SAFA) technology. We tested the feasibility of applying SAFA technology to create long-acting FSH as a therapeutic candidate for patients with hypogonadotropic hypogonadism. Methods: SAFA-FSH was produced using a Chinese hamster ovary expression system. To confirm the biological function, the production of cyclic AMP and phosphorylation of ERK and CREB were measured in TM4-FSHR cells. The effect of gonadotropin-releasing hormone agonists on spermatogenesis in a hypogonadal rat model was investigated. Results: In in vitro experiments, SAFA-FSH treatment increased the production of cyclic AMP and increased the phosphorylation of ERK and CREB in a dose-dependent manner. In animal experiments, sperm production was not restored by human chorionic gonadotropin treatment alone, but was restored after additional recombinant FSH treatment thrice per week or once every 5 days. Sperm production was restored even after additional SAFA-FSH treatment at intervals of once every 5 or 10 days. Discussion: Long-acting FSH with bioactivity was successfully created using SAFA technology. These data support further development of SAFA-FSH in a clinical setting, potentially representing an important advancement in the treatment of patients with hypogonadotropic hypogonadism.
Assuntos
Hormônio Foliculoestimulante , Hipogonadismo , Cricetinae , Humanos , Masculino , Ratos , Animais , Albumina Sérica , Células CHO , Cricetulus , Sêmen , Hipogonadismo/tratamento farmacológico , Gonadotropina Coriônica/uso terapêutico , Espermatogênese , Hormônio Foliculoestimulante Humano/uso terapêutico , Proteínas Recombinantes/uso terapêuticoRESUMO
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis around intrahepatic and extrahepatic bile ducts leading to severe hepatic cirrhosis and high mortality. Although there is an urgent clinical unmet need for PSC, no effective medical therapy has been developed to delay the disease progression until today. IL-18 binding protein (IL-18BP) is well-known to be a natural negative feedback regulator for IL-18, and we have developed a recombinant long-acting IL-18BP referred to as APB-R3 as a therapeutic agent to treat IL-18-related inflammatory diseases. Here, we aimed to study whether disrupted IL-18 signaling by APB-R3 treatment can inhibit PSC injuries in the experimental DDC diet-induced PSC rodent model. First, we found that the amounts of free IL-18 are augmented under PSC condition with increased expression of biliary IL-18 receptors. Administration of APB-R3 effectively attenuated key diagnostic parameters of PSC such as plasma ALP and GGT levels as well as bile acids levels. We also observed that blockade of IL-18 suppressed ductular reactive and proliferative phenotypes of cholangiocytes. Additionally, APB-R3 significantly ameliorated DDC diet-induced periductal fibrosis and transcriptional expressions of pro-fibrotic marker genes. Enhanced senescence associated secretory phenotype (SASP) markers in cholestatic liver disease were diminished by APB-R3 treatment. Our findings clearly demonstrate that the administration of IL-18BP biologics, APB-R3, effectively alleviates DDC diet-induced biliary injuries in rodent PSC model, implying APB-R3 can be a promising therapeutic reagent which warrants clinical human trials as new therapeutic options.
RESUMO
Off-label use of a human granulocyte colony stimulating factor (hG-CSF) has been allowed to treat dogs and cats with neutropenia. However, repeated administration of hG-CSF induces undesirable anti-drug antibody (ADA) responses, implying the necessity of animal-derived G-CSF as a therapeutic reagent, preferably with a long-acting capability. Herein, we generated a recombinant fusion protein by genetically combining FL335, a chimeric Fab specific for feline serum albumin (FSA), and feline G-CSF (fG-CSF), with the ultimate goal of developing a long-acting therapeutic fG-CSF for cats. The resulting FL335-fG-CSF fusion protein, referred to as APB-F1, was produced well as a functional form in a Chinese hamster ovary (CHO) expression system. In in vitro analyses, APB-F1 bound to FSA at high affinity (KD = 400 pM) and possessed 0.78 × 107 U/mg G-CSF biological activity, clearly proving its biological functionality. Pharmacokinetic (PK) and pharmacodynamic (PD) studies using healthy cats revealed that the serum half-life (t1/2) of APB-F1 was increased five times compared with that of fG-CSF (t1/2 = 13.3 h vs. 2.7 h) in subcutaneous (SC) injections. Additionally, APB-F1 induced a profound and sustained increase in white blood cell (WBC) and actual neutrophil count (ANC) up to 10 days, which was far superior to other G-CSF preparations, including filgrastim (Neupogen™) and even pegfilgrastim (Neulasta™). Conclusively, a long-acting fG-CSF with potent in vivo bioactivity was successfully created by using FL335; thus, we provided evidence that our "anti-serum albumin Fab-associated" (SAFA) technology can be applied reliably in developing valuable long-acting biologics in veterinary medicine.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Albumina Sérica/imunologia , Animais , Anticorpos , Células CHO , Doenças do Gato/terapia , Gatos , Cricetinae , Cricetulus , Cães , Fragmentos Fab das Imunoglobulinas/farmacologia , Neutropenia/terapia , Proteínas Recombinantes/farmacologiaRESUMO
Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (VH+CH1) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli.