Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning.
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.
Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Ave, Philadelphia, PA 19111, USA.
Qing-An
QA
Yuan
Matthew K
MK
Robinson
Heidi H
HH
Simmons
Maria
M
Russeva
Gregory P
GP
Adams
D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001435:Bacteriophages / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019151:Peptide Library; D018000:Receptors, Peptide / Q000276:immunology*; D018125:Receptors, Transforming Growth Factor beta / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D018000:Receptors, Peptide; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; C084621:anti-Mullerian hormone receptor