Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy Against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

Overview

Journal Toxins (Basel)

Publisher MDPI

Specialty Toxicology

Date 2019 Oct 17

PMID 31614771

Citations 7

Authors

Javier Ruiz-de-la-Herran

Jaime Tome-Amat

Rodrigo Lazaro-Gorines

Jose G Gavilanes

Javier Lacadena

Affiliations

Soon will be listed here.

Abstract

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.

Citing Articles

Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment.

Narbona J, Hernandez-Baraza L, Gordo R, Sanz L, Lacadena J Biomolecules. 2023; 13(7).

PMID: 37509078 PMC: 10377705. DOI: 10.3390/biom13071042.

A New Optimized Version of a Colorectal Cancer-Targeted Immunotoxin Based on a Non-Immunogenic Variant of the Ribotoxin α-Sarcin.

Narbona J, Gordo R, Tome-Amat J, Lacadena J Cancers (Basel). 2023; 15(4).

PMID: 36831456 PMC: 9954630. DOI: 10.3390/cancers15041114.

Internalization and trafficking of CSPG-bound recombinant VAR2CSA lectins in cancer cells.

Wang C, Nelepcu I, Hui D, Oo H, Truong S, Zhao S Sci Rep. 2022; 12(1):3075.

PMID: 35197518 PMC: 8866492. DOI: 10.1038/s41598-022-07025-6.

Antibody-Based Immunotoxins for Colorectal Cancer Therapy.

Sanz L, Ibanez-Perez R, Guerrero-Ochoa P, Lacadena J, Anel A Biomedicines. 2021; 9(11).

PMID: 34829955 PMC: 8615520. DOI: 10.3390/biomedicines9111729.

Dual Targeting of Cancer Cells with DARPin-Based Toxins for Overcoming Tumor Escape.

Shramova E, Proshkina G, Shipunova V, Ryabova A, Kamyshinsky R, Konevega A Cancers (Basel). 2020; 12(10).

PMID: 33081407 PMC: 7602955. DOI: 10.3390/cancers12103014.

References

Weldon J, Skarzynski M, Therres J, Ostovitz J, Zhou H, Kreitman R . Designing the furin-cleavable linker in recombinant immunotoxins based on Pseudomonas exotoxin A. Bioconjug Chem. 2015; 26(6):1120-8. PMC: 7724502. DOI: 10.1021/acs.bioconjchem.5b00190. View

Kimiz-Gebologlu I, Gulce-Iz S, Biray-Avci C . Monoclonal antibodies in cancer immunotherapy. Mol Biol Rep. 2018; 45(6):2935-2940. DOI: 10.1007/s11033-018-4427-x. View

Garcia-Ortega L, Masip M, Mancheno J, Onaderra M, Lizarbe M, Garcia-Mayoral M . Deletion of the NH2-terminal beta-hairpin of the ribotoxin alpha-sarcin produces a nontoxic but active ribonuclease. J Biol Chem. 2002; 277(21):18632-9. DOI: 10.1074/jbc.M200922200. View

del Pozo A, Gasset M, Onaderra M, Gavilanes J . Conformational study of the antitumor protein alpha-sarcin. Biochim Biophys Acta. 1988; 953(3):280-8. DOI: 10.1016/0167-4838(88)90036-2. View

De Lorenzo C, DAlessio G . From immunotoxins to immunoRNases. Curr Pharm Biotechnol. 2008; 9(3):210-4. DOI: 10.2174/138920108784567254. View

J M, Verma R . Therapeutic targets and recent advances in protein immunotoxins. Curr Opin Microbiol. 2012; 15(3):300-9. DOI: 10.1016/j.mib.2012.05.006. View

Santoro J, Campos-Olivas R, Lacadena J, del Pozo A, Gavilanes J, Rico M . The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity. J Mol Biol. 2000; 299(4):1061-73. DOI: 10.1006/jmbi.2000.3813. View

Tome-Amat J, Ruiz-de-la-Herran J, Martinez-Del-Pozo A, Gavilanes J, Lacadena J . α-sarcin and RNase T1 based immunoconjugates: the role of intracellular trafficking in cytotoxic efficiency. FEBS J. 2014; 282(4):673-84. DOI: 10.1111/febs.13169. View

Muyldermans S . Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013; 82:775-97. DOI: 10.1146/annurev-biochem-063011-092449. View

10.

Carreras-Sangra N, Tome-Amat J, Garcia-Ortega L, Batt C, Onaderra M, Martinez-Del-Pozo A . Production and characterization of a colon cancer-specific immunotoxin based on the fungal ribotoxin α-sarcin. Protein Eng Des Sel. 2012; 25(8):425-35. DOI: 10.1093/protein/gzs032. View

11.

Mattia A, Merker R . Regulation of probiotic substances as ingredients in foods: premarket approval or "generally recognized as safe" notification. Clin Infect Dis. 2008; 46 Suppl 2:S115-8. DOI: 10.1086/523329. View

12.

Garcia-Ortega L, Alegre-Cebollada J, Garcia-Linares S, Bruix M, Martinez-Del-Pozo A, Gavilanes J . The behavior of sea anemone actinoporins at the water-membrane interface. Biochim Biophys Acta. 2011; 1808(9):2275-88. DOI: 10.1016/j.bbamem.2011.05.012. View

13.

Olmo N, Turnay J, Gonzalez de Buitrago G, Lopez de Silanes I, Gavilanes J, Lizarbe M . Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis. Eur J Biochem. 2001; 268(7):2113-23. DOI: 10.1046/j.1432-1327.2001.02086.x. View

14.

Wawrzynczak E, Henry R, Cumber A, Parnell G, Derbyshire E, Ulbrich N . Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome-inactivating protein alpha-sarcin from Aspergillus giganteus. Eur J Biochem. 1991; 196(1):203-9. DOI: 10.1111/j.1432-1033.1991.tb15805.x. View

15.

Yoshida H . The ribonuclease T1 family. Methods Enzymol. 2001; 341:28-41. DOI: 10.1016/s0076-6879(01)41143-8. View

16.

LeMaistre C, Saleh M, Kuzel T, Foss F, Platanias L, Schwartz G . Phase I trial of a ligand fusion-protein (DAB389IL-2) in lymphomas expressing the receptor for interleukin-2. Blood. 1998; 91(2):399-405. View

17.

Mutter N, Soskine M, Huang G, Albuquerque I, Bernardes G, Maglia G . Modular Pore-Forming Immunotoxins with Caged Cytotoxicity Tailored by Directed Evolution. ACS Chem Biol. 2018; 13(11):3153-3160. PMC: 6243392. DOI: 10.1021/acschembio.8b00720. View

18.

Blanco-Toribio A, Lacadena J, Nunez-Prado N, Alvarez-Cienfuegos A, Villate M, Compte M . Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris. Microb Cell Fact. 2014; 13:116. PMC: 4249718. DOI: 10.1186/s12934-014-0116-1. View

19.

Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y . Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991; 266(26):17707-12. View

20.

OToole J, Esseltine D, Lynch T, LAMBERT J, Grossbard M . Clinical trials with blocked ricin immunotoxins. Curr Top Microbiol Immunol. 1998; 234:35-56. DOI: 10.1007/978-3-642-72153-3_3. View