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Gazzetta Medica Italiana Archivio per le Scienze Mediche 2017 May;176(5):299-307

DOI: 10.23736/S0393-3660.16.03370-2

Copyright © 2016 EDIZIONI MINERVA MEDICA

lingua: Inglese

Linker length effects in the conformation of M3 anti-CEA single chain Fv antibody fragment

Lincidio PÉREZ SÁNCHEZ, Monica BEQUET-ROMERO, Joem M. SERRANO, Hanssel BELL GARCIA, Marta AYALA AVILA, Leonardo CANAÁN-HADEN, Yanelys MORERA DIAZ, Jorge V. GAVILONDO

Recombinant Antibodies Laboratory, Cancer Department, Center of Genetic Engineering and Biotechnology (CIGB), Havana, Cuba


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BACKGROUND: Single-chain fragment variable (scFv) antibodies continue used as popular format of engineered antibodies. They have gained substantial clinical interest by their use as vehicles to deliver diagnostic and therapeutic agents such as radionuclides, enzymes, or toxins, selectively to tumor target cells. In this paper we study the relationship between linker length, multimerization behaviors and antigen binding activity of single chain Fv antibody fragment from CB/iorCEA.1 Mab name M3.
METHODS: Three variants of linkers were used: without linker (L0), linker of 5 amino acids (L5) and linker with 14 amino acids (L14). All these scFv antibodies fragments were produced in functional form in the periplasma of Escherichia coli and were purified by affinity and size exclusion chromatography.
RESULTS: Our results showed that antibody fragments M3 have a tendency to form trimer, dimer or monomer molecular species when the linker length is reduced. We also present a direct comparison of these molecules in terms of their in vitro targeting of human colon tumor cells LS 174T.
CONCLUSIONS: Our final observations indicate that in the case of this antibody dimeric species are likely to be obtained when a 5 amino acid linker is used and a pH of 7.5 is maintained during the whole process.


KEY WORDS: Carcinoembryonic antigen - Neoplasms - Single-chain antibodies - Periplasm - Escherichia coli - Imidazoleacetic acid

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