Affinity enhancement bivalent morpholino for pretargeting: initial evidence by surface plasmon resonance.
Pretargeting with bivalent effectors capable of bridging antitumor antibodies has been reported to provide superior results by affinity enhancement. Morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, likely to be a determinant of binding, may be adjusted simply by lengthening the chain. The goal of this investigation was to synthesize a bivalent MORF and to determine by surface plasmon resonance (SPR) whether the bivalent MORF exhibited bimolecular binding and whether the MORFs showed improved in vitro hybridization affinity in its bivalent form compared to its monovalent form. An 18 mer amino-derivitized MORF was made bivalent by dimerizing with disuccinimidyl suberate (DSS) in 1-methyl-2-pyrrolidinone (NMP) with N,N-diisopropylethylamine (DIEA) followed by purification by ion exchange chromatography. The in vitro hybridization affinity of bivalent compared to monovalent MORF was then measured by SPR. For these measurements, the complementary biotinylated cDNA was immobilized at coating densities that provided an average spacing of 20-100 angstroms and used to investigate the influence of this spacing on binding of the bivalent MORF with its binding regions separated by 25 A. The yield of bivalent MORF was as high as 45%, and the structure was confirmed by MALDI-TOF mass spectroscopy. When the sensograms obtained by SPR were analyzed using different binding models, the evidence was consistent with bimolecular binding of the bivalent MORF. The dissociation rate constant of the bivalent compared to monovalent MORF was more than 10-fold lower at 2.14 compared to 0.27 x 10(-5) (1/s) (p < 0.05), and since the association rate constants were similar at 8.53 and 5.64 x 10(5) (1/M.s) (p = 0.08), the equilibrium constant for hybridization to the immobilized cDNA of the bivalent compared to the monovalent MORF was almost 20-fold higher at 3.99 compared to 0.21 x 10(10) (1/M) (p < 0.05). In addition, qualitative evidence for bivalent binding of the bivalent MORF was apparent in the lower concentrations necessary to saturate the cDNA. Finally, the stoichiometry interpretation of the binding data provided estimates of the fraction of bivalent MORF binding bimolecularly. Under one set of conditions, this value was 20%. In conclusion, a bivalent MORF was easily synthesized by dimerization of a monovalent MORF. A lower dissociation rate constant and higher equilibrium constant was measured by SPR for the bivalent compared to monovalent MORF in their binding to an immobilized cDNA. These results show that bimolecular binding was occurring in the case of the bivalent MORF and suggest that bivalency may be superior to monovalency in MORF pretargeting applications.
He
Jiang
J
Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA. jiang.he@umassmed.edu
Liu
Guozheng
G
Vanderheyden
Jean-Luc
JL
Dou
Shuping
S
Mary
Rusckoswki
R
Hnatowich
Donald J
DJ
D000588:Amines; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D016373:DNA, Antisense / Q000138:chemical synthesis*; D018076:DNA, Complementary / Q000378:metabolism; D016503:Drug Delivery Systems; D015394:Molecular Structure; D009025:Morpholines; D060172:Morpholinos; D009693:Nucleic Acid Hybridization; D020349:Surface Plasmon Resonance
D000588:Amines; D003432:Cross-Linking Reagents; D016373:DNA, Antisense; D018076:DNA, Complementary; D009025:Morpholines; D060172:Morpholinos