Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers.
BACKGROUND
Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo.


OBJECTIVE
To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model.


METHODS
ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts.


RESULTS
125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h.


CONCLUSIONS
Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.
Wu
A M
AM
Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Chen
W
W
Raubitschek
A
A
Williams
L E
LE
Neumaier
M
M
Fischer
R
R
Hu
S Z
SZ
Odom-Maryon
T
T
Wong
J Y
JY
Shively
J E
JE
D000818:Animals; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D002277:Carcinoma / Q000276:immunology* / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013058:Mass Spectrometry; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D002272:Carcinoembryonic Antigen; D007128:Immunoglobulin Fragments; D010455:Peptides; C055966:immunoglobulin Fv