Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.
OBJECTIVE
To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors.


METHODS
hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In.


RESULTS
Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection.


CONCLUSIONS
The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.
Rossi
Edmund A
EA
IBC Pharmaceuticals, Inc., Morris Plains, Jew Jersey, USA.
Chang
Chien-Hsing
CH
Losman
Michele J
MJ
Sharkey
Robert M
RM
Karacay
Habibe
H
McBride
William
W
Cardillo
Thomas M
TM
Hansen
Hans J
HJ
Qu
Zhengxing
Z
Horak
Ivan D
ID
Goldenberg
David M
DM
D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry; D002272:Carcinoembryonic Antigen / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D002845:Chromatography; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006241:Haptens / Q000737:chemistry; D007205:Indium Radioisotopes / Q000737:chemistry; D007525:Isoelectric Focusing; D051379:Mice; D008819:Mice, Nude; D009101:Multiple Myeloma / Q000378:metabolism; D009368:Neoplasm Transplantation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D016719:Radioimmunodetection / Q000379:methods; D011868:Radioisotopes / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D018033:Antibodies, Bispecific; D002272:Carcinoembryonic Antigen; D006241:Haptens; D007205:Indium Radioisotopes; D010455:Peptides; D011868:Radioisotopes; D011993:Recombinant Fusion Proteins