Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from yeast.
BACKGROUND
The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody - directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.


RESULTS
An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.


CONCLUSIONS
The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.
Mallano
Alessandra
A
Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy. alessandra.mallano@iss.it
Zamboni
Silvia
S
Carpinelli
Giulia
G
Santoro
Filippo
F
Flego
Michela
M
Ascione
Alessandro
A
Gellini
Mara
M
Tombesi
Marina
M
Podo
Franca
F
Cianfriglia
Maurizio
M
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D043525:Cytosine Deaminase / Q000235:genetics / Q000276:immunology*; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D043525:Cytosine Deaminase
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