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

Olivia Cabanillas-Bernal,Salvador Dueñas,Marta Ayala-Avila,Teresa Escalante,Alexei F Licea-Navarro,Alexandra Rucavado,Paulo Lee Ho

doi:10.1371/journal.pone.0213394

Olivia Cabanillas-Bernal, Salvador Dueñas + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0213394

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Abstract

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.

Highlights

Due to the important role of antibodies in the survival, neutralization and elimination of antigens, intensive efforts have been focused on understanding and exploiting their molecular specificity
Each heavy chain contains five constant domains and a variable domain designated as variable domain of New Antigen Receptors (vNAR), which is responsible for antigen recognition [6]
A different type of vNAR than those reported previously has been identified in the immune repertoire of the horn shark (Heterodontus francisci), and to our knowledge, in this work we present the first description of this vNAR antibody

Summary

Introduction

Due to the important role of antibodies in the survival, neutralization and elimination of antigens, intensive efforts have been focused on understanding and exploiting their molecular specificity. With the objective of enhancing the properties of these molecules, different formats of antibodies have been developed, including Fab-fragments and single-chain Fv antibody fragments These molecules are heterodimers with variable heavy-chain (VH) and variable light-chain (VL) domains. In response to the tendency of reducing antibody formats to minimal fragments that retain their capacity for antigen union, two unique antibody isotypes have been found naturally containing a single domain for antigen binding. One of these antibody isoforms is found in the blood serum of Camelidae, these have been named heavy chain antibodies (HCAbs). Each heavy chain contains five constant domains and a variable domain designated as vNAR, which is responsible for antigen recognition [6]

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