Use of phage display to isolate specific human monoclonal antibody fragments against a potential target for multiple myeloma.
BACKGROUND
Multiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.


METHODS
Here, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.


RESULTS
Anti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.


CONCLUSIONS
These studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.
Liew
Pei Xiong
PX
Multiple Myeloma Research Laboratory, Singapore General Hospital, Singapore.
Ge
Feng
F
Gullo
Charles
C
Teoh
Gerrard K H
GK
Hwang
William Y K
WY
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000915:Antibody Affinity; D002460:Cell Line; D004265:DNA Helicases / Q000276:immunology*; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000276:immunology / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification*; D000072200:Ku Autoantigen; D009101:Multiple Myeloma / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen