Focà, Annalia
(2015)
Monoclonal antibodies as powerful tools in diagnosis and therapy.
[Tesi di dottorato]
| Tipologia del documento: |
Tesi di dottorato
|
| Lingua: |
English |
| Titolo: |
Monoclonal antibodies as powerful tools in diagnosis and therapy |
| Autori: |
| Autore | Email |
|---|
| Focà, Annalia | annalia.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: |
| nome | email |
|---|
| D'Auria, Maria Valeria | madauria@unina.it |
|
| Tutor: |
| nome | email |
|---|
| 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 |
[error in script]
[error in script]
| 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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