Panning phage antibody libraries on cells: isolation of human Fab fragments against ovarian carcinoma using guided selection.
The display of repertoires of human antibody (Ab) fragments on filamentous phages and selection by binding of the phage to antigen (Ag) have provided a ready means of deriving human Ab against purified Ag. However, it has been more difficult to obtain phage Ab against an individual Ag of a complex mixture, such as cell surface Ag. Using the technique of "guided selection," we generated human Ab against the high-affinity folate-binding protein (FBP), a cell surface Ag that is overexpressed in many human ovarian carcinomas. The guiding Ab template was provided by the light chain of mouse monoclonal Ab Mov19 (K[aff], 10[8] M[-1]) directed against FBP; this was paired with repertoires of human heavy chains displayed on phages, and the hybrid Ab fragments were selected by binding to an ovarian carcinoma cell line (OVCAR3). The selected human heavy chains were then paired with repertoires of human light chains. Further panning led to the isolation of a human Fab fragment, C4, with a binding affinity of 0.2 x 10(8) M(-1). This was highly specific for FBP, as demonstrated by ELISA and flow cytometry data and by immunoprecipitation of the relevant molecule from the cell surface of ovarian carcinoma cells. Moreover, C4 targeted the same or a closely related epitope of the Ag, as did the template rodent monoclonal Ab Mov19. These results suggest the usefulness of guided selection as a simple means to deriving human Ab against cell surface Ag for which a rodent Ab is available.
Figini
M
M
Istituto Nazionale per lo Studio e la Cura dei Tumori, Divisione di Oncologia Sperimentale E, Milan, Italy.
Obici
L
L
Mezzanzanica
D
D
Griffiths
A
A
Colnaghi
M I
MI
Winter
G
G
Canevari
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D002277:Carcinoma / Q000276:immunology*; D005260:Female; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000276:immunology*; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library