Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen.
The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.
Ravn
Peter
P
NEMOD Biotherapeutics GmbH & Co. KG, Robert-Rössle-Str 10, D-13125, Berlin-Buch, FRG, Germany.
Danielczyk
Antje
A
Jensen
Kim Bak
KB
Kristensen
Peter
P
Christensen
Peter Astrup
PA
Larsen
Martin
M
Karsten
Uwe
U
Goletz
Steffen
S
D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D002241:Carbohydrates / Q000276:immunology*; D002850:Chromatography, Gel; D005455:Fluorescent Antibody Technique; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification; D007700:Kinetics; D040081:Protein Array Analysis; D011485:Protein Binding / Q000276:immunology; D013997:Time Factors
D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D007135:Immunoglobulin Variable Region; C016467:Thomsen-Friedenreich antigen