Focà, Annalia (2015) Monoclonal antibodies as powerful tools in diagnosis and therapy. [Tesi di dottorato]

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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: Monoclonal antibodies as powerful tools in diagnosis and therapy
Autori:
AutoreEmail
Focà, Annaliaannalia.foca@unina.it
Data: 30 Marzo 2015
Numero di pagine: 162
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Farmacia
Scuola di dottorato: Scienze farmaceutiche
Dottorato: Scienza del farmaco
Ciclo di dottorato: 27
Coordinatore del Corso di dottorato:
nomeemail
D'Auria, Maria Valeriamadauria@unina.it
Tutor:
nomeemail
Iuvone, Teresa[non definito]
Sandomenico, Annamaria[non definito]
Data: 30 Marzo 2015
Numero di pagine: 162
Parole chiave: mAb, Nodal, Acetylated Lysines
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Depositato il: 10 Apr 2015 11:37
Ultima modifica: 28 Apr 2018 01:00
URI: http://www.fedoa.unina.it/id/eprint/10273
DOI: 10.6093/UNINA/FEDOA/10273

Abstract

STUDY I Nodal is a potent embryonic morphogen belonging to the TGF-beta superfamily. Typically, it binds to the Alk4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells and is absent in normal cells, including melanocytes. However it re-emerges in a number of human cancers, including melanoma, breast and colon cancer. Recent studies indicate that Nodal expression correlates with melanoma tumor progression toward a metastatic phenotype, indeed inhibition of the Nodal pathway also blocks the tumorigenic capacity and the plasticity of aggressive human melanoma cells. Recently, also Cripto-1 expression has been correlated to the pathogenesis and progression of human melanoma tumor. These findings suggest the hypothesis that inhibition of the Nodal-Cripto-1 signaling, which is supported by a direct binding between the two proteins, is a valid therapeutic approach against melanoma and increases the interest for Nodal as both a diagnostic or prognostic marker and as a potential new target for therapeutic intervention against melanoma. With the aim to produce molecules able to recognize Nodal and to block the signaling by preventing the association with Cripto-1, we have generated and screened a set of monoclonal antibodies targeting a major CBR (Cripto-Binding-Region) site which encompasses residues around Glu49 and Glu50 of human Nodal involved in the interaction with Cripto-1. With this approach, we have selected one, named 3D1, which strongly associates (KD about 1.4 nM) with full-length rhNodal. The selected mAb has been successfully tested for its ability to recognize endogenous Nodal protein in a panel of melanoma cell lines by both western blot and FACS analyses. Furthermore, the antibody inhibits the binding of Nodal to Cripto-1, as demonstrated by competitive SPR assays and, most importantly, blocks the Nodal-dependent activation of Smad2/3 in melanoma cancer cells. Notably, the antibody also blocks the Cripto-independent Nodal signalling converging on the MAPKs, as demonstrated by the concomitant effective blocking of MAPK activation. Given the autocrine mechanism of activation of Nodal (Smad2/3 regulate through Smad4 the expression of Nodal itself), the antibody also has the ability to reduce Nodal expression in the supernatants of C8161 cells. In 7 addition, it is also effective in reducing the C8161 clonogenicity and the typical vasculature-like phenotype induced by Nodal (vasculogenic mimicry). Remarkably, 3D1 in vivo strongly reduces lung colonization induced by C8161 metastatic cells, injected in mice. Moreover, immunohistochemical analyses revealed the ability of the 3D1 to affect the progression of the cell cycle by reducing Cyclin B1 and concurrently increasing p27 expression. The data accumulated on the 3D1 anti-Nodal mAb are all supportive of a huge diagnostic and therapeutic potential of this reagent. Next steps are therefore devoted to antibody affinity maturation to increase binding and selectivity and to humanization, to pave the way to in vivo experiments on human cells and tissues. STUDY II We have generated a set of monoclonal antibodies against the region 24-39 of human APE1/Ref1 protein, which is supposed to be multiple acetylated on lysines 27, 31, 32 and 35. In order to obtain mono- or multi-acetylated-APE1 specific antibodies, we have immunized mice with a peptides library, containing 16 different peptide antigens corresponding to all combinations of acetylated lysines and, after mAbs generation, we have screened the resulting clones to select those producing acetylation-specific mAbs. The screening, performed by ELISA, using isolated mono-, di-, tri-, and tetracetylated peptides, allowed to isolate four mAbs that bound with very high affinity and specificity to the acetylated peptides, while not recognizing the non acetylated variant. All selected antibodies displayed a poor selectivity against the differently acetylated peptides, being able to only discriminate between mono-acetylated/diacetylated/ tri-acetylated and tetracetylated variants. Furthemore, all selected mAbs were able to specifically recognize the acetylated form of the human APE1 protein, as revealed by Western blot analysis, while no detection was observed with the non-acetylated form and others acetylated proteins.

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