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Please use this identifier to cite or link to this item: http://hdl.handle.net/2108/700

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contributor.advisorCianfriglia, Maurizio-
contributor.authorZamboni, Silvia-
description20. cicloen
description.abstractThis study describes the genetic construction and expression of a fusion protein consisting of a human monoclonal antibody in a single chain fragment variable (scFv) format (E8) specific for a well defined determinant of the CEA cell surface antigen family and cytosine deaminase from yeast (yCD). DNA sequence encoding for the scFvE8 human monoclonal antibody recognizing an epitope shared by CEACAM1 (CD66) and CEACAM5 isoforms was assembled with a monomer of yCD. The scFvE8:yCD fusion protein possessed the binding specificity of the immuncompetent part of protein which include the recognition of melanoma (Mel P5) and colon carcinoma (LoVo) cell lines. The scFv8:yCD system showed the ability to make tumour cells naturally resistant to chemotherapy, susceptible to the non toxic substrate 5-fluorocytosine (5-FC) by its enzymatic conversion into 5-fluorouracil (5-FU). In vitro pre-treatment of Mel P5 and LoVo cell lines with scFvE8:yCD followed by cell washing and incubation with 5-FC, resulted in a significant cell killing supporting the utility of this fusion protein as an agent for selective tumor therapy by in loco prodrug activation. A convincing demonstration that such system can be developed for clinical use requires evidence that each of the components of the antibody complex functions by the mechanisms proposed This can be provided by well defined measurements including the concentration levels of the antibody-enzyme conjugate , in plasma, tumour and healthy tissues. To this aim we select from ETH-2 synthetic fagic library, a human monoclonal antibody in single chain fragment (scFv) format against a recombinant CD from yeast (yCD). This antibody proved to be functionally active in NMR and in in vitro studies to convert the antifungal drug 5-FC into the anticancer compound 5-FU. The specificity of the human scFv was confirmed by Western blot and ELISA analyses. With this antibody, yCD expression can now be monitored without interfering with its enzymatic function in ADEPT .en
description.tableofcontentsIntroduction - 1. Antibodies - 1.1 Antibodies as new therapeutic proteins - 1.2 Antibodies structure an function - 1.3 Conventional and Recombinant Monoclonal Antibodies - 1.4 Antibody Phage Display Technology - 1.4.1 Phage Display Technology - 1.4.2 Phage Display of Antibody Fragments - 1.4.3 Antibody Repertoires - 1.5 Engineered Antibody Fragments - 2 Immunotherapy - 2.1 Enhancing Effector Functions - 2.2 Direct Arming - 2.3 Indirect Arming - 2.4 Pretargeting Prodrug. - 3 ADEPT (Antibody Direct Enzyme Prodrug Therapy) - 3.1 Enzyme in ADEPT - 3.2 Antibodies in ADEPT - 3.3 Prodrugs in ADEPT - 3.4 Cytosine Deaminase - 3.5 5-Fluorouracil. - 4 Carcinoembrionic antigen (CEA) - 4.1 CEA structure - 4.2 CEA and CEA family genes - 4.3 CEA as tumor marker. - Materials and methods - Bacterial Strains - Cell lines - Antibodies and reagents - Primers - Genetic engineered constructs - Expression and Purification - SDS PAGE and WB analysis - ELISA - CEA binding specifity of the scFvE8:yCD - Functional Assay of the scFvE8:yCD on cells - Vector construction - Expression and Purification 2 - NMR - ETH-2 library - Selection of yCD protein specific antibody from ETH-2 library - ELISA 2 - DNA characterization and sequencing - Soluble scFv purification - SDS PAGE and WB analysis 2 - Determination of yCD activity - Cytotoxic assay. - Results - Genetic engineering of the scFv antibodies for ADEPT - The antigen specificity of the engineered fusion proteins - Functional assay of the scFvE8:yCD - Construction of scFv specific to yCD - Expression and purification of yCD protein - Selection and characterization of scFvH5 antibody specific to yCD - Determination of yCD activity - Cytotoxic assay. - Discussion - Referencesen
format.extent2387245 bytes-
format.extent31288 bytes-
subject.classificationBIO/11 Biologia molecolareen
titleDesign of human single chain fragments for antibody direct enzyme prodrug therapy (ADEPT)en
typeDoctoral thesisen
degree.nameDottorato in microbiologia medica e immunologiaen
degree.disciplineFacoltà di medicina e chirurgiaen
degree.grantorUniversità degli studi di Roma Tor Vergataen
date.dateofdefenseA.A. 2007/2008en
Appears in Collections:Tesi di dottorato in medicina

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