Generation and testing of engineered multimeric Fabs of trastuzumab
2020
Selis, Fabio | Sandomenico, Annamaria | Cantile, Maria | Gianco, Ricky | Calvanese, Luisa | Falcigno, Lucia | Dell'Omo, Paolo | Esperti, Andrea | De Falco, Sandro | Focà, Annalia | Caporale, Andrea | Iaccarino, Emanuela | Truppo, Emanuela | Scaramuzza, Silvia | Tonon, Giancarlo | Ruvo, Menotti
Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules.Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells.Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody.Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.
Показать больше [+] Меньше [-]Ключевые слова АГРОВОК
Библиографическая информация
Эту запись предоставил National Agricultural Library