Single-chain antibody fragment-based adsorbent for the extracorporeal removal of beta2-microglobulin.
BACKGROUND
Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution.


METHODS
An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum.


RESULTS
The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m.


CONCLUSIONS
Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.
Grovender
Eric A
EA
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Kellogg
Brenda
B
Singh
Jasleen
J
Blom
Daniel
D
Ploegh
Hidde
H
Wittrup
K Dane
KD
Langer
Robert S
RS
Ameer
Guillermo A
GA
D000595:Amino Acid Sequence; D000686:Amyloidosis / Q000097:blood / Q000628:therapy*; D001483:Base Sequence; D003001:Cloning, Molecular; D005112:Extracorporeal Circulation; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007163:Immunosorbent Techniques; D007676:Kidney Failure, Chronic / Q000097:blood / Q000628:therapy*; D008969:Molecular Sequence Data; D010957:Plasmids; D015003:Yeasts / Q000235:genetics; D001613:beta 2-Microglobulin / Q000276:immunology* / Q000302:isolation & purification*
D007135:Immunoglobulin Variable Region; D001613:beta 2-Microglobulin