Research Article: Conformational characterization of a novel anti-HER2 candidate antibody

Date Published: May 9, 2019

Publisher: Public Library of Science

Author(s): Leina Moro Pérez, Azalia de la Caridad Rodríguez Taño, Lázaro Roberto Martín Márquez, Jose Alberto Gómez Pérez, Aisel Valle Garay, Rancés Blanco Santana, Sabato D’Auria.

http://doi.org/10.1371/journal.pone.0215442

Abstract

Regulatory agencies establish that a broad physicochemical and biological characterization is necessary for the evaluation of comparability between a biosimilar candidate product and a reference commercial drug. Between them, conformational characterization of proteins is of vital importance to determine its folding and biological functions. In this work, the conformational features of a novel monoclonal antibody (called 5G4) were evaluated by means of circular dichroism spectroscopy and fluorescence. Secondary structure and thermal stability of mAbs were determined by circular dichroism in the far ultraviolet, while three-dimensional folding of proteins was analyzed by both circular dichroism in the near ultraviolet and intrinsic tryptophan fluorescence. In all experiments, Herceptin (Roche) was used as control. Both antibodies showed a composition of secondary structure predominantly of β-sheets (55–56%) and thermal stability of ~ 75°C, suggesting structural similarity. The three-dimensional folding of proteins was also similar due to the absorption spectra of the aromatic residues and the emission wavelength maxima by fluorescence were comparable. The values of the fluorescence attenuation constant (Stern-Volmer constant) for increasing concentrations of acrylamide were also similar, suggesting a degree of exposure of tryptophan residues similar, although it was slightly decreased for Herceptin. Our data permit to consider that 5G4 monoclonal antibody showed similar conformational characteristics when compared with Herceptin.

Partial Text

Despite significant advances in the diagnosis and treatment of cancer, this disease remains one of the leading causes of morbidity and mortality in the world [1]. Breast cancer is the most common cause of cancer-related deaths in women, comprising almost a third of all malignancies in females [2]. The heterogeneous nature of breast cancer has implications for both patients and medical research. For this reason, treatment strategies are directed towards molecular markers [3]. Currently, one of the major advances in this therapy has been the introduction of monoclonal antibodies (mAbs) targeting specific molecules overexpressed during tumorigenesis (e.g. growth factor receptors) [4].

In the present work, it is shown a conformational characterization study of a novel anti-HER2 mAb (5G4) by means of CD and Trp intrinsic fluorescence. The analysis of the secondary structure by far-UV CD showed typical spectral characteristics of β-sheets for 5G4 mAb, similar to those previously reported for Herceptin [26]. This result was corroborated when the percentage of secondary structures for both mAbs was compared using the DichroWeb tool (55 to 56% of β-sheets). Moreover, the three-dimensional folding of 5G4 mAb showed the appearance of absorption bands corresponding to the aromatic residues. The intensity of the Trp absorption signal for this mAb was comparable to Herceptin, although small differences for the Tyr and Phe residues were observed. These results are in correspondence with obtained by other authors for Herceptin [9].

 

Source:

http://doi.org/10.1371/journal.pone.0215442

 

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