High-throughput generation and engineering of recombinant human antibodies.
The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.
Krebs
B
B
MorphoSys AG, Lena-Christ-Strasse 48, 82152 Martinsried, Germany.
Rauchenberger
R
R
Reiffert
S
S
Rothe
C
C
Tesar
M
M
Thomassen
E
E
Cao
M
M
Dreier
T
T
Fischer
D
D
Höss
A
A
Inge
L
L
Knappik
A
A
Marget
M
M
Pack
P
P
Meng
X Q
XQ
Schier
R
R
Söhlemann
P
P
Winter
J
J
Wölle
J
J
Kretzschmar
T
T
D000818:Animals; D000915:Antibody Affinity; D000917:Antibody Formation; D000951:Antigens, Neoplasm / Q000276:immunology; D001331:Automation; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D018922:HL-60 Cells; D015236:HLA-C Antigens / Q000276:immunology; D019073:HT29 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D016177:Macrophage-1 Antigen / Q000276:immunology; D019151:Peptide Library; D011233:Precipitin Tests / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D020349:Surface Plasmon Resonance
D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors; D015236:HLA-C Antigens; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018799:Intercellular Adhesion Molecule-1; D016177:Macrophage-1 Antigen; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C528994:HLA-C*06 antigen; C055966:immunoglobulin Fv