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1.
Front Endocrinol (Lausanne) ; 14: 1132172, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36909328

RESUMEN

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.


Asunto(s)
Hormona Folículo Estimulante , Hipogonadismo , Cricetinae , Humanos , Masculino , Ratas , Animales , Albúmina Sérica , Células CHO , Cricetulus , Semen , Hipogonadismo/tratamiento farmacológico , Gonadotropina Coriónica/uso terapéutico , Espermatogénesis , Hormona Folículo Estimulante Humana/uso terapéutico , Proteínas Recombinantes/uso terapéutico
2.
Vet Immunol Immunopathol ; 240: 110322, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34509747

RESUMEN

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.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , Albúmina Sérica/inmunología , Animales , Anticuerpos , Células CHO , Enfermedades de los Gatos/terapia , Gatos , Cricetinae , Cricetulus , Perros , Fragmentos Fab de Inmunoglobulinas/farmacología , Neutropenia/terapia , Proteínas Recombinantes/farmacología
3.
Immunol Lett ; 207: 46-55, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30684504

RESUMEN

Recombinant human interferon beta (rIFN-ß) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-ß is desirable since only one pegylated version of long-acting rIFN-ß-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-®, we generated a fusion construct by linking human IFN-ß cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-ß-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/ß receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-ß-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-ß-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-ß-1a is worthy of further development as an alternative long-acting IFN-ß therapeutic.


Asunto(s)
Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoterapia/métodos , Interferón beta/metabolismo , Esclerosis Múltiple/terapia , Proteínas Recombinantes de Fusión/metabolismo , Animales , Células CHO , Cromatografía , Cricetulus , Tasa de Filtración Glomerular , Semivida , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Interferón beta-1a/farmacocinética , Interferón beta-1a/uso terapéutico , Interferón beta/genética , Interferón beta/farmacocinética , Macaca fascicularis , Esclerosis Múltiple/inmunología , Unión Proteica , Estabilidad Proteica , Ratas , Ratas Endogámicas Lew , Receptor de Interferón alfa y beta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Albúmina Sérica/inmunología , Albúmina Sérica/metabolismo
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