Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.
The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.
Rauchenberger
Robert
R
MorphoSys AG, Lena-Christ-Strasse 48, 82152 Martinsried, Germany.
Borges
Eric
E
Thomassen-Wolf
Elisabeth
E
Rom
Eran
E
Adar
Rivka
R
Yaniv
Yael
Y
Malka
Michael
M
Chumakov
Irina
I
Kotzer
Sarit
S
Resnitzky
Dalia
D
Knappik
Achim
A
Reiffert
Silke
S
Prassler
Josef
J
Jury
Karin
K
Waldherr
Dirk
D
Bauer
Susanne
S
Kretzschmar
Titus
T
Yayon
Avner
A
Rothe
Christine
C
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001667:Binding, Competitive; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D003001:Cloning, Molecular; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D051379:Mice; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature; D013997:Time Factors
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008024:Ligands; D019151:Peptide Library; D020033:Protein Isoforms; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C496365:FGFR3 protein, human; C496366:Fgfr3 protein, mouse; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3