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)
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)
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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 To circumvent some of these problems, toxins have been replaced by mammalian RNases, molecules per se not cytotoxic, which become toxic when internalized by target cells.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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