Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display

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Abstract

The recognition molecule L1 plays important functional roles in the nervous system and in non-neural tissues. Since antibodies to L1 are of prime importance to study its functional properties, we have generated affinity matured human single chain variable fragment (scFv) antibodies against mouse L1 by introducing random mutations in the complementarity determining regions (CDRs) of a previously isolated scFv antibody heavy chain (CDR1 and CDR2) and light chain (CDR3). After biopanning the mutant library, a clone (5F7) that gave the strongest ELISA signal was expressed, purified, and characterized. The dissociation constant of 5F7 (2.86×10−8 M) was decreased 60-fold compared to the wild type clone G6 (1.72×10−6 M). 5F7 detected L1 by Western blot analysis in mouse brain homogenates and recognized L1 in L1 transfected cells and cryosections from mouse retina and optic nerve by immunofluorescence. Bivalent 5F7 scFv antibody (5F7-Cys) was also generated and showed a dissociation constant of 5.22×10−9 M that is 5.5-fold lower than that of monomeric 5F7 antibody. The bivalent affinity matured L1 scFv antibody thus showed stronger binding by a factor of 310 compared to the wild type clone. This antibody should be useful in various biological assays.

Section snippets

Materials and methods

Design and construction of a mutant library for L1 scFv antibody affinity maturation and cloning. For the construction of a L1 scFv affinity matured library, the gene of L1 scFv clone G6 selected from a human synthetic phage display scFv library was used as template for PCR amplification. Oligonucleotides used for PCR amplification of heavy and light chain V-regions genes are described in Table 1. The amino acid residues of the antibody are numbered according to Kabat and colleagues [16]. The

Construction of mutant phage display scFv library for affinity maturation of L1 scFv antibody

In a previous study [15], we have isolated a scFv antibody (G6, GenBank Accession No.: AF 394236) using the human synthetic phage display scFv library [29]. This antibody bears moderate affinity (Kd 2×10−6 M) and reacted specifically with mouse L1 by ELISA and Western blot analysis under non-reducing conditions. However, the antibody did not react with L1 by Western blot analysis under reducing conditions, or by indirect immunohistology on fresh frozen sections of adult mouse brain.

We therefore

Discussion

We have been successful in producing a second generation of single chain variable fragment (scFv) antibodies against the murine neural cell adhesion molecule L1 by site-directed random mutagenesis. We generated a library that contains mutations in the three complementarity determining regions (CDRs) of the scFv heavy chains (CDR1 and CDR2) and the light chain (CDR3) of a previously isolated low affinity binding scFv antibody against mouse L1 [15]. Introduction of mutations in the CDRs is a

Acknowledgements

We are grateful to Dr. Greg Winter for providing the synthetic human phage display scFv library, Dr. Stefan Dubel for providing the pOPE101-215(Yol) vector, and Dr. Dario Neri for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (Scha 185/18-1,2).

References (41)

  • F. Hardy et al.

    Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay

    J. Immunol. Methods

    (1997)
  • D. Neri et al.

    Biophysical methods for the determination of antibody–antigen affinities

    Trends Biotechnol.

    (1996)
  • A. Schmiedl et al.

    Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli

    J. Immunol. Methods

    (2000)
  • R.E. Hawkins et al.

    The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme

    J. Mol. Biol.

    (1993)
  • W.P. Yang et al.

    CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range

    J. Mol. Biol.

    (1995)
  • R. Schier et al.

    Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site

    J. Mol. Biol.

    (1996)
  • M. Moos et al.

    Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin

    Nature

    (1988)
  • P. Hulley et al.

    L1 neural cell adhesion molecule is a survival factor for fetal dopaminergic neurons

    J. Neurosci. Res.

    (1998)
  • S. Chen et al.

    Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1

    J. Neurobiol.

    (1999)
  • M. Dahme et al.

    Disruption of the mouse L1 gene leads to malformations of the nervous system

    Nat. Genet.

    (1997)
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