Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein

Sivaccumar, Jwala P.; Iaccarino, Emanuela; Oliver, Angela; Cantile, Maria; Olimpieri, Pierpaolo; Leonardi, Antonio; Ruvo, Menotti; Sandomenico, Annamaria

doi:10.3390/ph16081130

Open AccessArticle

Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein

by

Jwala P. Sivaccumar

^1,†,

Emanuela Iaccarino

¹

,

Angela Oliver

^1,2,

Maria Cantile

³,

Pierpaolo Olimpieri

⁴,

Antonio Leonardi

⁵

,

Menotti Ruvo

^1,*

and

Annamaria Sandomenico

^1,*

¹

Institute of Biostructures and Bioimaging, CNR, Via P. Castellino, 111, 80131 Naples, Italy

²

Università degli Studi della Campania Luigi Vanvitelli, Via Vivaldi 43, 81100 Caserta, Italy

³

BIOVIIIX, via A. Manzoni, 1, 80123 Naples, Italy

⁴

Department of Physics, Sapienza University, 00184 Rome, Italy

⁵

Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, via Pansini 5, 80131 Naples, Italy

^*

Authors to whom correspondence should be addressed.

^†

Present address: Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.

Pharmaceuticals 2023, 16(8), 1130; https://doi.org/10.3390/ph16081130

Submission received: 29 June 2023 / Revised: 29 July 2023 / Accepted: 2 August 2023 / Published: 10 August 2023

(This article belongs to the Section Biopharmaceuticals)

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Abstract

Drug development in recent years is increasingly focused on developing personalized treatments based on blocking molecules selective for therapeutic targets specifically present in individual patients. In this perspective, the specificity of therapeutic targets and blocking agents plays a crucial role. Monoclonal antibodies (mAbs) and their surrogates are increasingly used in this context thanks to their ability to bind therapeutic targets and to inhibit their activity or to transport bioactive molecules into the compartments in which the targets are expressed. Small antibody-like molecules, such as Fabs, are often used in certain clinical settings where small size and better tissue penetration are required. In the wake of this research trend, we developed a murine mAb (3D1) neutralizing the activity of Nodal, an oncofetal protein that is attracting an ever-increasing interest as a selective therapeutic target for several cancer types. Here, we report the preparation of a recombinant Fab of 3D1 that has been humanized through a computational approach starting from the sequence of the murine antibody. The Fab has been expressed in bacterial cells (1 mg/L bacterial culture), biochemically characterized in terms of stability and binding properties by circular dichroism and bio-layer interferometry techniques and tested in vitro on Nodal-positive cancer cells.

Keywords: Nodal; recombinant Fab; humanization

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MDPI and ACS Style

Sivaccumar, J.P.; Iaccarino, E.; Oliver, A.; Cantile, M.; Olimpieri, P.; Leonardi, A.; Ruvo, M.; Sandomenico, A. Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein. Pharmaceuticals 2023, 16, 1130. https://doi.org/10.3390/ph16081130

AMA Style

Sivaccumar JP, Iaccarino E, Oliver A, Cantile M, Olimpieri P, Leonardi A, Ruvo M, Sandomenico A. Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein. Pharmaceuticals. 2023; 16(8):1130. https://doi.org/10.3390/ph16081130

Chicago/Turabian Style

Sivaccumar, Jwala P., Emanuela Iaccarino, Angela Oliver, Maria Cantile, Pierpaolo Olimpieri, Antonio Leonardi, Menotti Ruvo, and Annamaria Sandomenico. 2023. "Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein" Pharmaceuticals 16, no. 8: 1130. https://doi.org/10.3390/ph16081130

APA Style

Sivaccumar, J. P., Iaccarino, E., Oliver, A., Cantile, M., Olimpieri, P., Leonardi, A., Ruvo, M., & Sandomenico, A. (2023). Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein. Pharmaceuticals, 16(8), 1130. https://doi.org/10.3390/ph16081130

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Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein

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