Design of human single chain fragments for antibody direct enzyme prodrug therapy (ADEPT)

UNIVERSITÀ DEGLI STUDI DI ROMA

"TOR VERGATA"

FACOLTA' DI MEDICINA E CHIRURGIA

DOTTORATO DI RICERCA IN

IMMUNOLOGIA E MICROBIOLOGIA MEDICA

XX Ciclo

Design of Human Single Chain Fragments for Antibody Direct

Enzyme Prodrug Therapy (ADEPT)

SILVIA ZAMBONI

Docente Guida: Prof. Maurizio Cianfriglia

Coordinatore: Prof. Carlo Federico Perno

TABLE OF CONTENTS

ABSTRACT………

INTRODUCTION………

1 ANTIBODIES

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1.1 Antibodies as new therapeutic proteins……….

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1.2 Antibodies structure an function

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1.3 Conventional and Recombinant Monoclonal Antibodies

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1.4 Antibody Phage Display Technology

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1.4.1 Phage Display Technology

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1.4.2 Phage Display of Antibody Fragments

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1.4.3 Antibody Repertoires

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1.5 Engineered Antibody Fragments

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2 IMMUNOTHERAPY

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2.1 Enhancing Effector Functions

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2.2 Direct Arming

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2.3 Indirect Arming

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2.4 Pretargeting Prodrug

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3 ADEPT (Antibody Direct Enzyme Prodrug Therapy)

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3.1 Enzyme in ADEPT

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3.3 Prodrugs in ADEPT

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3.4 Cytosine Deaminase

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3.5 5-Fluorouracil

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4 CARCINOEMBRIONIC ANTIGEN (CEA)

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4.1 CEA structure

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4.2 CEA and CEA family genes

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4.3 CEA as tumor marker

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MATERIALS AND METHODS………

Bacterial Strains

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Cell lines

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Antibodies and reagents

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Primers

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Genetic engineered constructs

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Expression and Purification 1

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SDS PAGE and WB analysis 1

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ELISA 1

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CEA binding specifity of the scFvE8:yCD

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Functional Assay of the scFvE8:yCD on cells

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Vector construction

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Expression and Purification 2

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ETH-2 library

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Selection of yCD protein specific antibody from ETH-2 library

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ELISA 2

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DNA characterization and sequencing

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Soluble scFv purification

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SDS PAGE and WB analysis 2

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Determination of yCD activity

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Cytotoxic assay

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RESULTS………

Genetic engineering of the scFv antibodies for ADEPT

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The antigen specificity of the engineered fusion proteins

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Functional assay of the scFvE8:yCD

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Construction of scFv specific to yCD

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Expression and purification of yCD protein

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Selection and characterization of scFvH5 antibody specific to yCD

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Determination of yCD activity

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Cytotoxic assay

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DISCUSSION………..