[Frontiers in Bioscience 11, 1733-1749, May 1, 2006]

Optimal design of Ig 5' primers for construction of diverse phage antibody library established to select anti-HAb18GEF and anti-DOTA-Y Fabs for hepatoma pretargeting RIT

Sihe Zhang 1, Jinliang Xing 1, Qing Zhang 2, Fei Song 1, Yu Li 1, Xiangmin Yang 1, and Zhinan Chen 1

1Cell Engineer Research Center, State Key Laboratory of Cancer Biology, 2 Department of Clinical Labortory, Tangdu Hospital, Fourth Military Medical University, Xi`an 710032, P.R. China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Cells, vectors, bacterial strains, and reagents
3.2. Design of murine Fd 5' primers and Lκ 5' primers
3.3. Site-directed mutagenesis of pComb3 phagemid vector
3.4. Evaluation of murine Fd (Lκ) 5' primers and cloning of Ig gene
3.5. Construction and identification of bacterial-form Fab libraries
3.6. Preparation and biopanning of phage display Fab libraries
3.7. Selection of different antigen-binding phage antibodies (PhAbs)
3.8. Soluble Fab expression, characterization and sequence analysis
4. Results
4.1. Design of murine Fd 5' primers and Lκ 5' primers
4.2. Evaluation of murine Fd 5' primers and Lκ 5' primers
4.3. Identification of constructed Fab antibody libraries
4.4. Biopanning of Fab phage libraries and selection of different antigen-specific PhAbs
4.5. Soluble Fab expression and characterization
5. Discussion
6. Acknowledgement
6. References

1. ABSTRACT

Phage antibody library yields antibodies with higher affinity against different antigens, if diverse IgV gene repertoires can be amplified. As the currently available Fab primer sets cannot guarantee efficient amplification with high diversity, and because rare cloning sites can be found in certain Ig genes, here, we present an optimal set of Ig 5' primers, compatible with Fd 5' clone site replaced pComb3 vector, for diverse Fab phage display library construction. These novel Fab primes designed based on the newly classified IgV families, not only have best match and highest coverage for IgV family with minimized N-terminal amino acid changes, but also present good amplification diversity and efficiency of Ig gene from mice immunized with different forms of antigens (HAb18GEF, KLH-DOTA-Y, and HAb18G/pcDNA3). A high quality immune phage library with good diversity was constructed based on the mixed Ig repertoire, and five high affinity Fab antibodies were selected to specifically bind to HAb18GEF, DOTA-Y and an irrelevant antigen γ-sm, respectively. This novel Fab primers set can be applied to the construction of diverse phage antibody library and the anti-HAb18GEF and anti-DOTA-Y Fab antibodies lay a solid foundation for radioimmunotherapy of hepatoma.