Download The cDNA fragments which encode the variable regions of the light

Generation of recombinant ligands to human prostate cancer
cell as innovative tools for diagnosis and therapy.
A single chain antibody (scFv) was constructed from a hybridoma expressing the anti-PMSA
monoclonal antibody CB-PSMA and expressed in E. Coli TG1.
Prostate cancer is the most frequent malignancy in the male population of western countries and the
second cause of death in men after cardiovascular disease. Today immunotherapeutic approach is
considered very promising to diagnose and treat different kinds of cancer; so the development of
new generation of reagents with improved features is necessary to support or to replace monoclonal
antibodies produced by hybridoma technology. During the development of a panel of antibodies to
prostate cancer-specific antigens, CB-PSMA was selected, a monoclonal antibody to Prostate
Specific Membrane Antigen. PSMA is a type 2 transmembrane glycoprotein discovered in the
androgen dependent LNCaP human prostatic adenocarcinoma cell line; in healthy subjects its
expression is prostate specific and it has been shown to be up regulated in poorly differentiated,
metastatic and hormone-refractory carcinomas. Therefore PSMA has great potential as diagnostic
and therapeutic tool in the managment of advanced prostate cancer, and it is a very attractive target
for mAb-based imaging. To allow repeated dosing of antibodies in patients is necessary to decrease
their immunogenicity, so we started CB-PSMA humanization by murine CDR grafting into human
frameworks to retain binding properties of murine mAb and improve its tolerability.
The first step to antibodies humanization by CDR grafting consists in isolating CDR regions on
antibody variable fragments.
We amplified variable regions (VH and VL) from hybridoma mRNA using primers containing a
mixture of sequences designed to hybridize to VH and VL framework regions conserved between
antibody subfamily members. We then fused VH and VL by addition of a (Gly4 Ser)3 linker by
Splicing Overlap Extension PCR and expressed the scFv in E. Coli TG1.
However, a decrease in the binding affinity of scFv compared to the parental Mab has been
observed. With many scFv molecules, this decrease in the affinity results when the format is
switched from the larger bivalent monoclonal antibody to the smaller monovalent scFv. Strategies
to overcome this problem includes affinity maturation and size and valence modification.
Site-directed mutagenesis with degenerated primers and phage-display selection contribute a valid
method for ricombinant antibody affinity maturation.
We have constructed a page-display library of scFv CBsc-PSMA mutants with high variability,
introducing random mutations at positions 31-32-33 in CDR1 and 50-52-52a-54 in CDR2 of VH
nucleotide sequence. These residues are found to frequently contact the antigen in the known threedimensional structures of antibody-antigen complexes. The resulting repertoire clones were selected
for binding to PMSA-transfected cells. After three rounds of panning, and screening of many
individual clones, antibodies with improved affinity were isolated.
The creation of scFv antibody allowed us to confirm rightness and reactivity of isolated variable
regions as well as to dispose of an useful intermediate product for CB-PSMA humanization. It has
been shown that single chain antibodies use can be advantageous for certain applications as in in
vivo imaging, infact their small structure (nearly 30kDa rather than 150kDa of IgG) promotes
tissues penetration and speeds up clearance time. ScFv murine antibodies could be tested also as
therapeutic tools because of their potential reduced immunogenicity