Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.
We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.
Adamson
P J
PJ
Child Health Research Institute, Women's and Children's Hospital, Leukocyte Biology Laboratory, 72 King William Road, North Adelaide, SA 5006, Australia.
Millard
D J
DJ
Hohmann
A W
AW
Mavrangelos
C
C
Macardle
P J
PJ
Pilkington
G
G
Mulhern
T D
TD
Tedder
T F
TF
Zola
H
H
Nicholson
I C
IC
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions / Q000235:genetics*; D018951:Antigens, CD20 / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000235:genetics*; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region; D008958:Models, Molecular; D009154:Mutation
D018951:Antigens, CD20; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region