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1.
Int J Mol Sci ; 18(10)2017 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-28953230

RESUMO

Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 108 clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Anticorpos de Cadeia Única/farmacologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Biblioteca de Peptídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Prep Biochem Biotechnol ; 47(6): 619-626, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28151045

RESUMO

Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Glycine max/genética , Hirudinas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Soja/genética , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Hirudinas/metabolismo , Humanos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Soja/metabolismo , Glycine max/metabolismo
3.
Biomedicines ; 9(12)2021 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-34944600

RESUMO

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10-5 s-1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

4.
EBioMedicine ; 65: 103250, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33647772

RESUMO

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favourable druggability by utilizing phage display-based strategy. METHODS: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. FINDINGS: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10-6 s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P = 0.658, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively. INTERPRETATION: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.


Assuntos
Anticorpos/imunologia , Peptídeos/metabolismo , Pró-Proteína Convertase 9/metabolismo , Animais , Anticorpos/uso terapêutico , Reações Antígeno-Anticorpo , LDL-Colesterol/sangue , Células Hep G2 , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/patologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Ligação Proteica , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Regulação para Cima/efeitos dos fármacos
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