Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides

Citation
A. Knappik et al., Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides, J MOL BIOL, 296(1), 2000, pp. 57-86
Citations number
118
Language
INGLESE
art.tipo
Review
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
0022-2836 → ACNP
Volume
296
Issue
1
Year of publication
2000
Pages
57 - 86
Database
ISI
SICI code
0022-2836(20000211)296:1<57:FSHCAL>2.0.ZU;2-P
Abstract
By analyzing the human antibody repertoire in terms of structure, amino aci d sequence diversity and germline usage, we found that seven V-H and seven V-L (four V kappa and three V lambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived fo r each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for div ersification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking th e CDRs. Molecular modeling verified that all canonical classes were present . We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries t otalling 2 x 10(9) members was created after cloning the genes in all 49 co mbinations into a phagemid vector, itself devoid of the restriction sites i n question. Diversity was created by replacing the V-H and V-L CDR3 regions of the master genes by CDR3 Library cassettes, generated from mixed trinuc leotides and biased towards natural human antibody CDR3 sequences. The sequ encing of 257 members of the unselected libraries indicated that the freque ncy of correct and thus potentially functional sequences was 61 %. Selectio n experiments against many antigens yielded a diverse set of binders with h igh affinities. Due to the modular design of all master genes, either singl e binders or even pools of binders can now be rapidly optimized without kno wledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be in corporated quickly, and the separation of the frameworks may help in analyz ing why nature has evolved these distinct subfamilies of antibody germline genes. (C) 2000 Academic Press.