       Document 0661
 DOCN  M9630661
 TI    CDR walking mutagenesis for the affinity maturation of a potent human
       anti-HIV-1 antibody into the picomolar range.
 DT    9603
 AU    Yang WP; Green K; Pinz-Sweeney S; Briones AT; Burton DR; Barbas CF 3rd;
       Department of Molecular Biology, Scripps Research Institute, La; Jolla,
       CA 92037, USA.
 SO    J Mol Biol. 1995 Dec 1;254(3):392-403. Unique Identifier : AIDSLINE
       MED/96095799
 AB    We describe the investigation of methodologies for the creation of very
       high affinity human antibodies. The high affinity human antibody b4/12
       was optimized for its affinity to the human envelope glycoprotein gp120
       of human immunodeficiency virus type 1 (HIV-1). Five libraries of b4/12
       were constructed by saturation mutagenesis of
       complementarity-determining regions (CDRs). Libraries of antibody Fab
       fragments were displayed on the surface of filamentous phage and
       selected in vitro for binding to immobilized gp120. Sequential and
       parallel optimization strategies of CDRs were examined. The sequential
       CDR walking strategy consistently yielded b4/12 variants of improved
       affinity in each of the four different optimization sequences examined.
       This resulted in a 96-fold improvement in affinity. Additivity effects
       in the antibody combining site were explored by combining independently
       optimized CDRs in the parallel optimization strategy. Six variants
       containing optimized CDRs were constructed. Improvement of affinity
       based on additivity effects proved to be unpredictable but did lead to a
       modest improvement in affinity. Indeed, only one of the six combinations
       demonstrated additivity. The highest affinity Fab prepared using this
       strategy was improved 420-fold in affinity. The affinity of this Fab was
       15 pM as compared to 6.3 nM for b4/12. Examination of the kinetics of
       Fab binding to gp120 revealed that improvements in affinity were
       dominated by a slowing of the off-rate of the Fab. The methodology
       presented here provides a route for the improvement of the affinities of
       antibodies typical of tertiary immune responses into the picomolar
       range. Such improvements may have profound effects on the utility of
       antibodies as therapeutic and prophylactic agents.
 DE    Amino Acid Sequence  Antibody Affinity/*GENETICS  Antigen-Antibody
       Reactions  Bacteriophages/GENETICS  Base Sequence  Binding Sites,
       Antibody/*GENETICS  Comparative Study  Gene Library  HIV
       Antibodies/*GENETICS/IMMUNOLOGY  HIV Envelope Protein gp120/*IMMUNOLOGY
       HIV-1/*IMMUNOLOGY  Immunoglobulins, Fab/GENETICS  Immunoglobulins,
       Heavy-Chain/GENETICS  Immunoglobulins, Light-Chain/GENETICS  Models,
       Molecular  Molecular Sequence Data  *Mutagenesis  Support, Non-U.S.
       Gov't  Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

