<i>In silico-designed</i> mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization

Millán-Gómez, Dalia; Dueñas, Salvador; Muñoz, P.; Camacho-Villegas, Tanya A.; Elosua, Carolina; Cabanillas-Bernal, Olivia; Escalante, Teresa; Perona, Almudena; Abia, David; Drescher, Florian; Fournier, Pierrick G.J.; Ramos, Marco A.; Mares, Rosa E.; Paniagua-Solis, Jorge; Mata-Gonzalez, Teresa; González-Canudas, Jorge; Hoffman, Robert M.; Licea-Navarro, Alexei F.; Sánchez-Campos, Noemí

doi:10.18632/oncotarget.25549

Cited by 4 publications

(8 citation statements)

References 34 publications

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“…The in vitro anti-angiogenic effect of VS1-20 and VS0-4 was evaluated and results suggest that these antibodies are capable of inhibiting angiogenesis in a three-dimensional model based on endothelial cell spheroids. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival vNARs whose affinity had been improved through in silico modeling, with in vitro and in vivo anti-angiogenic effect previously reported [24]. This study also presents the first report of the construction of synthetic libraries of vNAR domains, using frameworks with three different number of Cys at their CDR3.…”

Section: Introductionsupporting

confidence: 51%

“…Docking, protein-protein affinity and interaction surface predictions of anti-VEGF antibodies, suggested that VS1-20 and VS0-4 recognize different epitopes, whereas VS1-20 and V13_P98Y recognize very close epitopes in the target molecule. The In silico analysis suggested that VS1-20 has a greater interaction with VEGF than VS0-4 and even than the control antibody V13_P98Y, an in silico maturated antibody with an in vitro and in vivo anti-angiogenic effect previously reported [24]. The results of the in silico analysis were supported by a competitive ELISA, it was evaluated if VS1-20 and V13_P98Y have an overlapping epitope and could block the union each other to VEGF.…”

Section: Discussionmentioning

confidence: 67%

“…A comparative analysis of the interaction energy of each model was performed (Table 3). In the control model V13_P98Y [24], an interaction score of -38.94 REU was identified. The CDR3 loop of V13_P98Y has the high score of interaction in the complex (Fig 9A and 9B).…”

Section: Resultsmentioning

confidence: 99%

“…To evaluate whether the antibodies isolated from the synthetic libraries were functional, the antibodies against VEGF were selected. VEGF is a model antigen in our research group and to date anti-VEGF antibodies have been isolated from immune libraries [12] and antibodies have been developed whose VEGF neutralization has been improved through in silico modeling [24].…”

Section: Discussionmentioning

confidence: 99%

“…For a better interpretation of our results we use a positive control (V13_P98Y/VEGF) [24] to have an idea of the REU values obtained for the interaction with an in silico , in vitro an in vivo developed VNAR against VEGF.…”

Section: Methodsmentioning

confidence: 99%

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Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3

et al. 2019

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The variable domain of New Antigen Receptors (vNAR) from sharks, present special characteristics in comparison to the conventional antibody molecules such as: small size (12–15 kDa), thermal and chemical stability and great tissue penetration, that makes them a good alternative source as therapeutic or diagnostic agents. Therefore, it is essential to improve techniques used for the development and selection of vNAR antibodies that recognize distinct antigens. The development of synthetic antibody libraries offers a fast option for the generation of antibodies with the desired characteristics. In this work three synthetic antibody libraries were constructed; without cysteines (Cys), with one Cys and with two Cys residues within its CDR3, with the objective of determining whether the presence or absence of Cys in the CDR3 favors the isolation of vNAR clones from a synthetic library. The libraries were validated selecting against six mammalian proteins. At least one vNAR was found for each of the antigens, and a clone coming from the library without Cys in the CDR3 was selected with all the antigens. In vitro angiogenesis assay with the isolated anti-VEGF antibodies, suggest that these vNARs are capable of inhibiting in vitro angiogenesis. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival antibodies with affinity maturation by in silico modeling.

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Section: Introductionsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3

et al. 2019

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Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders

Gasperin-Bulbarela

Cabanillas-Bernal

Dueñas

et al. 2022

Methods in Molecular Biology

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Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability

Ubah¹,

Buschhaus²,

Ferguson³

et al. 2018

Biochemical Society Transactions

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Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150 kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel ‘antibody-like’ binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease.

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In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization

Cited by 4 publications

References 34 publications

Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3

Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3

Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders

Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability

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