Paciello, Rolando (2015) Human antitumor and antiviral Immunoagents. [Tesi di dottorato]

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Abstract

Immunotherapy is a precious strategy to fight cancer and viral infections. Phage display technology allows for the production of fully human immunoagents specific for tumor associated antigens (TAA) or for cell surface receptors involved in virus infection. By using this technology we isolated fully human antibody fragments (scFvs) specific for the extracellular loops of Claudin-1 (CLDN-1), a tight junction protein essential for hepatitis C virus (HCV) cell entry and following infection. The selected scFvs have been converted in human IgG4 antibodies and characterized. They show high selective binding affinities for CLDN-1-positive cells, recognize distinct epitopes of the protein, and specifically inhibit HCV infection in a dose-dependent manner in cell cultures of human hepatocytes. An attractive TAA for cancer immunotherapy is ErbB2, a tyrosine kinase receptor overexpressed on many different types of tumor cells, such as breast and gastric cancer, whereas it is expressed at low levels on normal cells and only on certain epithelial cell types. A human antibody fragment (scFv), named Erbicin, specific for ErbB2 receptor has been isolated in our laboratory by using phage display technology. Erbicin was fused with a human pancreatic RNase (HP-RNase) variant resistant to the cytosolic inhibitor, to obtain a novel anti-ErbB2 immunoRNase (IR), which was called Erb–HPDDADD-RNase. The novel IR binds with high affinity to a panel of ErbB2-positive gastric tumor cells and inhibits their growth more efficiently than the parental immunoRNase, which is not resistant to the inhibitor. Moreover, Erb–HP-DDADD-RNase is endowed with an antitumor activity for Herceptin-resistant cancer cells both in vitro and in vivo. Similar results were obtained fusing Erbicin with the Fc region of a human IgG1 to obtain a construct, which was called Erb-hcAb for its compact size (100 kDa) if compared with the full size (155 kDa) of a natural IgG. Here we report on the antitumor effects on gastric cancer cells of Erb-hcAb which targets a different epitope of ErbB2 with respect to trastuzumab (Herceptin) and pertuzumab (Omnitarg), the only anti-ErbB2 antibodies currently in clinical use for breast cancer therapy. The results demonstrate that the growth of gastric cancer cells is efficiently inhibited in vitro and in vivo by Erb-hcAb, which shows antitumor effects on the NCI-N87 cell line more potent than those observed for Herceptin. In conclusion, these human immunoagents may represent valuable tools for both antitumor and antiviral therapies in association or in alternative to conventional therapies currently used.

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