Abstract
We report the construction and the use of a phage display human antibody library (>3 x 108 clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 μg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 107-108 M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K(d) = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.
| Original language | English |
|---|---|
| Pages (from-to) | 21769-21776 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 273 |
| Issue number | 34 |
| DOIs | |
| Publication status | Published - Aug 21 1998 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Design and use of a phage display library : Antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. / Pini, Alessandro; Viti, Francesca; Santucci, Annalisa; Carnemolla, Barbara; Zardi, Luciano; Neri, Paolo; Neri, Dario.
In: Journal of Biological Chemistry, Vol. 273, No. 34, 21.08.1998, p. 21769-21776.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Design and use of a phage display library
T2 - Antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel
AU - Pini, Alessandro
AU - Viti, Francesca
AU - Santucci, Annalisa
AU - Carnemolla, Barbara
AU - Zardi, Luciano
AU - Neri, Paolo
AU - Neri, Dario
PY - 1998/8/21
Y1 - 1998/8/21
N2 - We report the construction and the use of a phage display human antibody library (>3 x 108 clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 μg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 107-108 M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K(d) = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.
AB - We report the construction and the use of a phage display human antibody library (>3 x 108 clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 μg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 107-108 M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K(d) = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.
UR - http://www.scopus.com/inward/record.url?scp=0032555478&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032555478&partnerID=8YFLogxK
U2 - 10.1074/jbc.273.34.21769
DO - 10.1074/jbc.273.34.21769
M3 - Article
C2 - 9705314
AN - SCOPUS:0032555478
VL - 273
SP - 21769
EP - 21776
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 34
ER -