ANALYSIS OF A NUCLEIC-ACID-BINDING ANTIBODY FRAGMENT - CONSTRUCTION AND CHARACTERIZATION OF HEAVY-CHAIN COMPLEMENTARITY-DETERMINING REGION SWITCH VARIANTS
Mj. Calcutt et al., ANALYSIS OF A NUCLEIC-ACID-BINDING ANTIBODY FRAGMENT - CONSTRUCTION AND CHARACTERIZATION OF HEAVY-CHAIN COMPLEMENTARITY-DETERMINING REGION SWITCH VARIANTS, Gene, 168(1), 1996, pp. 9-14
The display of antibody (Ab) fragments (Fab) on the surface of filamen
tous bacteriophage (phage) and selection of phage that interact with a
particular antigen (Ag) has enabled the isolation of Fab that bind nu
cleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective
tissue disease patients and certain inbred strains of mice and are tho
ught to be pathogenic. Although there is ample data concerning the ami
no acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA,
significantly less is known about how autoAb interact with NA. The co
mplementarity-determining regions (CDR) contained in the Fab contribut
e the most to Ag binding, especially through heavy (H)-chain CDR 3. We
have examined the role of individual H-chain CDR of a previously isol
ated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic ac
id interaction using a combination of H-chain CDR switching and soluti
on-binding experiments. The three H-chain CDR of DNA-1 Fab were indepe
ndently switched with the H-chain CDR of a Fab (D5) with very similar
sequence and framework (FR) that binds DNA poorly in order to create a
ll possible H-chain CDR combinations. The chimeric Fab genes were bact
erially expressed, and their products were purified and analyzed. Resu
lts indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of t
he remainder of the H-chain of D5 Fab, restored binding to oligo(dT)(1
5) to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with C
DR 2 of D5 Fab restored binding to 100%. A combination of H-chain CDR
2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted i
n the ability of the chimeric Fab to bind RNA preferentially over DNA.
These studies demonstrate the importance of both H-chain CDR 1 and 3
in DNA recognition and further suggest that the specificity of the typ
e of NA recognized by a particular Fab can be drastically altered by e
xchanging CDR.