In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer.
The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.
N. V. Organon, Oss, The Netherlands.
E S
ES
Bos
W H
WH
Kuijpers
M
M
Meesters-Winters
D T
DT
Pham
A S
AS
de Haan
A M
AM
van Doornmalen
F M
FM
Kaspersen
C A
CA
van Boeckel
F
F
Gougeon-Bertrand
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000276:immunology; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007075:Immunoglobulin M / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D016499:Radioimmunotherapy / Q000379:methods*
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D009841:Oligonucleotides