Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice

Kosztyu, Petr; Kuchař, Milan; Černý, Jiří; Barkocziová, Lucia; Malý, Michal; Petroková, Hana; Czerneková, Lýdie; Lišková, Veronika; Kafková, Leona Rašková; Knotigová, Pavlína Turánek; Mašek, Josef; Tuřánek, Jaroslav; Malý, Petr; Raška, Milan

doi:10.1016/j.ebiom.2019.07.015

Cited by 9 publications

(25 citation statements)

References 59 publications

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“…The most widely used scaffolds are designed Ankyrin repeats (dARPins), lipocalin domain (anticalins), and Ig-binding domain of Staphylococcal Protein A (affibodies) [ 17 , 18 , 19 ]. Some of the small protein scaffolds were already proven to be useful in a wide range of applications, from academic research to clinical imaging, diagnostics, or therapeutics [ 20 , 21 , 22 , 23 ], and others are even in clinical trials [ 24 , 25 ]. In our previous work, we used the albumin-binding domain (ABD) of Streptococcal Protein G as a scaffold protein.…”

Section: Introductionmentioning

confidence: 99%

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Pham

Huličiak

Biedermannová

et al. 2021

Viruses

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Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

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Section: Introductionmentioning

confidence: 99%

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Pham

Huličiak

Biedermannová

et al. 2021

Viruses

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show abstract

“…We have also previously developed a human mAb targeting the C-terminal domain (CTD) of PCSK9 utilizing phage display-based strategy [ 29 ]. Molecular modeling and docking techniques are widely used to predict the binding mode of a drug with its protein target [ 68 , 69 ].…”

Section: Discussionmentioning

confidence: 99%

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Bai

Mei

et al. 2021

Biomedicines

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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.

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“…Molecular Modeling and docking techniques are wildly used to predict the binding mode of an antibody with its protein target (Kosztyu et al 2019, Vahed et al 2020. In this work, the docking results (Fig.…”

Section: Hypolipidemic Effect Of H5e12-l230g In Mice Over-expressing ...mentioning

confidence: 99%

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis

Bai

Mei

et al. 2021

Preprint

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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. To eliminate the cytotoxic effector functions and mitigate the heterogeneity, the heavy-chain constant region of h5E12-L230G was modified with L234A/L235A/N297G mutations and C-terminal lysine deletion. 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 reduced the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to Alirocumab. Our data suggest that h5E12-L230G is a highly potent antibody binding to PCSK9 catalytic domain with slow dissociation rate which may be utilized as a therapeutic candidate for treating hypercholesterolemia and relevant cardiovascular diseases.

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Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice

Cited by 9 publications

References 59 publications

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis

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