Biochemical and Biophysical Research Communications
Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display
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)
The L1 family of neural cell adhesion molecules: old proteins performing new tricks
Neuron
(1996)Neural recognition molecules and synaptic plasticity
Curr. Opin. Cell Biol.
(1997)- et al.
Errors in corticospinal axon guidance in mice lacking the neural cell adhesion molecule L1
Curr. Biol.
(1998) - et al.
Mutations in the cell adhesion molecule L1 cause mental retardation
Trends Neurosci.
(1995) - et al.
Role of L1 in neural development: what the knockouts tell us
Mol. Cell. Neurosci.
(1998) - et al.
Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations
Curr. Opin. Neurobiol.
(1998) - et al.
Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel
J. Biol. Chem.
(1998) - et al.
Canonical structures for the hypervariable regions of immunoglobulins
J. Mol. Biol.
(1987) - et al.
By-passing immunization. Human antibodies from V-gene libraries displayed on phage
J. Mol. Biol.
(1991) - et al.
Measurements of the true affinity constant in solution of antigen–antibody complexes by enzyme-linked immunosorbent assay
J. Immunol. Methods
(1985)
Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay
J. Immunol. Methods
Biophysical methods for the determination of antibody–antigen affinities
Trends Biotechnol.
Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli
J. Immunol. Methods
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.
CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range
J. Mol. Biol.
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.
Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin
Nature
L1 neural cell adhesion molecule is a survival factor for fetal dopaminergic neurons
J. Neurosci. Res.
Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1
J. Neurobiol.
Disruption of the mouse L1 gene leads to malformations of the nervous system
Nat. Genet.
Cited by (18)
PD-1 /PD-L1 checkpoint in hematological malignancies
2018, Leukemia ResearchCitation Excerpt :Aim of this review is to revise the medical literature regarding the role of PD-1 checkpoint in principal hematologic neoplastic disorders and the future therapeutic perspective offered by its blockade. The PD-1/PD-L1 axis is one of the major mechanisms of immune escaping exerted by several cancer types in which upregulation of PD-L1 is observed [1–3]. PD-L1 interacts with its inhibitory receptor PD-1 expressed on activated T cells with the function of promoting self-antigen tolerance and balances the immune regulation to avoid antigen persistence and immune-mediated pathologies [4,5] The PD-L1 immune induced expression by tumor is considered to be an adaptive resistance mechanism for tumor cells in response to immune challenge [3,6].
A 104kDa Aedes aegypti aminopeptidase N is a putative receptor for the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis
2013, Insect Biochemistry and Molecular BiologyCan IgE-mediated allergic diseases be prevented by using allergen-specific IgG antibodies?
2011, Medical HypothesesCitation Excerpt :In antibody molecules, the complementarity-determining regions (CDRs) especially CDR3 of the heavy- and light-chains, are responsible for high-affinity binding with antigens. It is possible to mature affinity by chain shuffling or site-directed mutagenesis in recombinant antibody technology to improve affinity of the original antibody [19,20]. The strength of antibody binding to its antigen is not only determined by affinity but also by the valency of interaction, with multivalent interactions leading to an increase in functional affinity.
Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin
2009, Insect Biochemistry and Molecular BiologyBacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae
2007, Journal of Biological ChemistryCitation Excerpt :Table 1 shows that all Cry1Ab mutants bound the Bt-R1 fragment with a similar dissociation constant as wild-type Cry1Ab. This ELISA procedure permits the determination of true association rate constants in solution, and several reports have shown good agreement in the determination of KD values by ELISA binding competition assays and those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, fluorescence transfer, or surface plasmon resonance) (35–37, 45). Toxicity Directly Correlates with in Vitro Oligomer Formation and Pore Formation—The Cry1Ab mutants were proteolytically activated in the presence of antibody scFv73 to determine if the mutations affected the formation of pre-pore oligomeric structure.