Arciello, Angela
(2005)
Construction and characterization of a fully human
antitumor immunoRNase selective for ErbB2-positive carcinomas: analysis of the role of the membrane transporter ABCC2/MRP2 in multidrug
resistance (MDR).
[Tesi di dottorato]
(Inedito)
| Tipologia del documento: |
Tesi di dottorato
|
| Lingua: |
English |
| Titolo: |
Construction and characterization of a fully human
antitumor immunoRNase selective for ErbB2-positive carcinomas: analysis of the role of the membrane transporter ABCC2/MRP2 in multidrug
resistance (MDR) |
| Autori: |
| Autore | Email |
|---|
| Arciello, Angela | [non definito] |
|
| Data: |
2005 |
| Tipo di data: |
Pubblicazione |
| Numero di pagine: |
181 |
| Istituzione: |
Università degli Studi di Napoli Federico II |
| Dipartimento: |
Chimica biologica |
| Dottorato: |
Oncologia ed endocrinologia molecolare |
| Ciclo di dottorato: |
16 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| Vecchio, Giancarlo | [non definito] |
|
| Tutor: |
| nome | email |
|---|
| Piccoli, Renata | [non definito] |
|
| Data: |
2005 |
| Numero di pagine: |
181 |
| Settori scientifico-disciplinari del MIUR: |
Area 06 - Scienze mediche > MED/06 - Oncologia medica |
[error in script]
[error in script]
| Depositato il: |
28 Set 2005 |
| Ultima modifica: |
01 Dic 2014 13:43 |
| URI: |
http://www.fedoa.unina.it/id/eprint/98 |
| DOI: |
10.6092/UNINA/FEDOA/98 |
Abstract
The aim of the study was the construction and characterization of a novel,
fully human immunoRNase (IR). IRs, chimeric proteins made up of a ribonuclease
(RNase) molecule fused to an antibody moiety, represent a valid alternative to
immunotoxins (ITs). ITs directed to cell surface molecular targets have shown to
have a therapeutic potential. However, they also have limitations, mainly
represented by nonspecific toxicity and by the immunogenicity of their bacterial or
plant toxins. To circumvent some of these problems, toxins have been replaced by
mammalian RNases, molecules per se not cytotoxic, which become toxic when
internalized by target cells. In particular, the use of human RNases,
physiologically present in extracellular fluids and tissues, has allowed the
construction of immunoconjugates potentially less immunogenic. Obviously, the
preparation of fully human IRs is highly desiderable to obtain effective and tumorselective,
but also immunocompatible immunoagents.
A novel, fully human IR was prepared in the present study. It is made up
of human pancreas RNase (HP-RNase), fused to a human single chain variable
fragment (scFv) directed to the ErbB2 receptor. This scFv, named Erbicin, has
been isolated in our laboratory from a large human scFv phagemid library. ErbB2,
a tyrosine kinase receptor, represents one of the most specific tumor associated
antigens (TAA), since this receptor is overexpressed in clinically significant
tumors, such as breast, ovary and lung carcinomas. On the other hand, in normal
tissues it is expressed only in certain epithelial cell types.
The novel, fully human anti-ErbB2 IR, named hERB-hRNase, was
successfully expressed in bacterial cells and isolated from the periplasmic fraction.
The characterization of its biological properties showed that the human IR retains
the enzymatic activity of the wild-type ribonuclease (HP-RNase) and specifically
binds to ErbB2-positive cells with high affinity, as the parental scFv. Furthermore,
our studies showed that the human IR undergoes receptor-mediated endocytosis in
target cells. In fact, the IR behaves as an immunoprotoxin and upon internalization
by target cells becomes selectively cytotoxic in a dose-dependent manner at
nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice
bearing an ErbB2-positive tumor, hERB-hRNase induced a dramatic reduction of
tumor volume.
The immunoconjugate hERB-hRNase is the first fully human antitumor
IR produced so far, with a high potential as a poorly immunogenic human drug
devoid of nonspecific toxicity, directed against ErbB2-positive malignancies.
The ABC (ATP binding cassette) family of membrane transport proteins
includes the best known mediators of resistance to anticancer drugs, such as the
ABC transporters ABCB1 (MDR1/P-gp), ABCC1/MRP1, and ABCG2/MXR.
However, although ABC transporters overexpression appears to be a major cause
of failure in the treatment of cancer, acquired resistance to multiple anticancer
drugs may also be multifactorial, involving alteration of detoxification processes,
apoptosis, DNA repair and drug uptake.
The human prostate cancer cell line RC0.1 has been derived from the
parental sensitive cell line DU145 by continuous exposure to the drug rubitecan
(9-nitro-camptothecin), an inhibitor of topoisomerase I, now in phase III clinical
trials. Several factors have been proposed to explain the phenotype of
camptothecin resistance in RC0.1 cell line. Microarray analyses, performed using a
novel ABC-ToxChip, indicated the overexpression of the ABC transporter MRP2
in the resistant cell line RC0.1. In order to establish the role of MRP2 in
camptothecin resistance, different strategies were used in the present study to
inhibit this ABC transporter. The inhibition of MRP2 expression was obtained by
RNA interference technology, whereas the inhibition of its biological activity was
achieved using the inhibitor MK571. In both cases, the resistance phenotype of
RC0.1 tumor cells was not reverted. This indicated that MRP2 does not play a
crucial role in the resistance mechanism. To explain MRP2 overexpression, we
hypothesized that this represents an early adaptation of the cells to the drug,
providing the background for the evolution of different resistance mechanisms.
The expression profile of the 48 ABC transporters has been characterized
in a panel of 60 diverse cancer cell lines (the NCI-60), used by the National
Cancer Institute (NCI) to screen for anticancer activity. By correlating the
expression results with the growth inhibitory profiles of 1,429 candidate anticancer
drugs tested against the cells, transporters able to confer resistance to anticancer
drugs have been identified. In particular, 28 compounds were predicted to be less
active in cells that expressed large amounts of MRP2. Such a prediction has been
confirmed in the present study. Furthermore, the analysis of the chemical
structures of the 28 identified MRP2 substrates led to the identification of common
structural features, such as a cyclopentanone ring, an element structurally related
to the cyclopentenone ring of prostaglandins. This observation led us to
hypothesize a new role for MRP2 transporter, such as its involvement in the
transport of cyclopentenone prostaglandins. Prostaglandins are involved in a wide
variety of physiological and pathophysiological processes, but the mechanism of
prostaglandin release from cells is not completely understood. Here we
demonstrate an effective involvement of MRP2 in the transport of the
cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2).
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