Generation of a polyclonal fab phage display library to the human breast carcinoma cell line BT-20.
We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.
Santora
K E
KE
Department of Pathology and Laboratory Medicine, and the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, Boston, MA 02118, USA.
Sarantopoulos
S
S
Den
W
W
Petersen-Mahrt
S
S
Sompuram
S R
SR
Sharon
J
J
D000818:Animals; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments