Avidin-biotin system pretargeting radioimmunoimaging and radioimmunotherapy and its application in mouse model of human colon carcinoma.

AIM: To evaluate the multi-step pretargeting radioimmunoimaging (RII) and radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma with avidin-biotin system labeled with (153)Sm. METHODS: Two- and three-step strategies for avidin-biotin system pretargeting techniques were established. In a three-step procedure, human colon carcinoma bearing nude mice were first injected with biotinylated monoclonal antibody (McAb-Bt) followed by cold avidin (Av) 48 h later and then (153)Sm-DB(2) 24 h thereafter; whereas the two-step procedure consisted of injection of (153)Sm-SA 48 h after pretargeting with biotinylated anti-CEA monoclonal antibody (CEA McAb-Bt). SPECT imaging and biodistribution were performed at 4, 24, 48, or 72 h after injection of (153)Sm-labeled compounds. Five groups of nude mice subcutaneously grafted with human colon carcinoma were treated 3 d after grafting. One group received the injection with 100 microg CEA McAb-Bt followed by cold avidin (80 microg) after 2 d and 11.1 MBq (153)Sm-DB(2) after 1 d. Four control groups were treated respectively with 11.1 MBq (153)Sm-CEA McAb, 11.1 MBq (153)Sm-nmIgG, 11.1 MBq (153)Sm-DB(2), 100 microL normal saline. Toxicity was evaluated by changes of leukocyte count, and the efficacy by variation in tumor volume. Histological analyses of tumors were performed. RESULTS: The three-step procedure allowed faster blood clearance and yielded higher tumor blood ratios (5.76 at 4 h and 12.94 at 24 h) of the (153)Sm-DB(2). The tumor was clearly visualized at 4 h in gamma-imaging after the injection of (153)Sm-DB(2), while a significant accumulation of (153)Sm-SA in the tumor was observed only 24 h after the injection and tumor blood ratios at 4 and 24 h were 1.00 and 2.03, respectively, in the two-step procedure. Pretargeting RIT and (153)Sm-CEA McAb had a strong tumor-inhibiting effect. The tumor inhibitory rate was 80.67% and 78.44%, respectively, five weeks after therapy. Histopathological evidence also indicated radioactive damage in tumor tissues as necrosis of tumor cells, while in the other organs such as liver and kidney no radioactive damage was observed. Leukocyte counts showed significant decrease after treatment in groups of (153)Sm-CEA McAb and (153)Sm-nmIgG. CONCLUSION: The two kinds of pretargeting strategies can elevate the target-to-nontarget ratio, decrease the blood background and shorten the imaging time compared to (153)Sm-CEA McAb. Three-step pretargeting RIT is as efficient as (153)Sm-CEA McAb, but markedly less toxic. This study provides experimental evidence for the clinical application of pretargeting RII and RIT.

Radioimmunotherapy with 153Sm-CEA monoclonal antibody in nude mice bearing human colon carcinoma: an experimental study.

OBJECTIVE: To observe the therapeutic effect of 153Sm-labeled CEA monodonal antibody(mAb) in nude mice bearing human colon carcinoma. METHODS: Fifteen nude mice were subjected to subcutaneous inoculation of human colon carcinoma cells, and 3 days later, they were divided into 3 groups with equal numbers to receive single high-dose injection of 11.1 MBq 153Sm-CEA mAb (therapy group), 11.1MBq 153SmCl3 (therapy control group), or 100 microl normal saline (non-treatment control group). The tumor-inhibiting effect of 153Sm-labeled CEA mAb was evaluated in terms of body weight changes and tumor volume variation 4 weeks after the treatment. Histological analysis of tumors were performed in all the groups after the all other observations were completed. RESULTS: 153Sm-CEA mAb had a significant anti-tumor effect, with a tumor inhibition rate of 74.29% at 4 weeks after treatment, while for 153SmCl3, the inhibition rate was only 15.90%. Rapid tumor growth was observed in non-treatment control group. No significant difference in the body weight changes was noted between the 3 groups. Histopathological examination revealed tumor necrosis as the evidence for radioactive damage in therapy group, which was not observed in non-treatment control group. CONCLUSION: 153Sm-CEA mAb has a strong selective inhibitory effect against colon carcinoma and may be potentially used as an agent in radioimmunotherapy.

Fused Expression of Anti-CEA Single-chain Antibody Gene with Core-streptavidin Gene in Insect Cells.

A fused construct formed by an anti-CEA single-chain antibody gene with a core-streptavidin gene(ScFv-CS)was inserted into the donor plasmids pFastBacHTa and expressed in Tn-5B1-4 cells. SDS-PAGE analysis revealed that the molecular weight expressed product of ScFv-CS were 41 kD. Using horseradish peroxidase(HRP)labeled biotin as antibody product Western blot, one band of 41 kD only was detected. It was also shown by RIA that the expression product possessed the ability to bind to its specific antigen of CEA. These results showed that the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.

Fusion and Expression of the Genes Encoding Murine-Human Chimeric Heavy Chain to Carcinoembryonic Antigen with Core-streptavidin Gene.

In order to reduce the human anti-murine antibody (HAMA) in radioimmunotherapy, the gene encoding the heavy chain variable region (V(H)) of the murine monoclonal antibody with high specificity and affinity to carcinoembryonic antigen was fused to the human C(gamma3) gene to construct the murine-human chimeric heavy chain antibody gene, then was linked to core-streptavidin which can specifically bind to biotin, facilitating its purification and radioisotope labeling. The fusion gene was expressed in E.coli at high level, accounting for 24% of the total bacteria protein, and was characterized by SDS-PAGE and Western-blots. When using RIA the content of inclusion bodies was denatured and followed by renaturation, it was shown by using RIA to possess ability to bind to its specific antigen of CEA. Using horseradish peroxidase (HRP) labeled biotin as antibody in Western-blots, one band of 70 kD only was detected, demonstrating the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.

Expression of a Gene for Single-chain Antibody to Carcinoembryonic Antigen in Bombyx mori cells and Its Larvae.

Recombinant Bm-BacScFv virus which contains a cDNA encoding anti-CEA single-chain antibody was generated by cotransfection into Bm N cells with the transfer plasmid pBacPAK-(His)(6)-ScFv and the modified Bombyx mori nuclear polyhedrosis virus Bm-BacPAK genomic DNA. Bombyx mori cells and larvae were infected by the recombinant virus, respectively. The anti-CEA single-chain antibody were expressed both in Bombyx mori cells and larvae, the former accounted for 6% of the total cell protein, the later 0.3 mg per larvae. The histidine-tagged ScFvs were respectively purified with nickel-iminodiacetic acid affinity chromatography. The purity of the former reached over 90% and that of the latter was lower. Purified protein products was able to bind to CEA with an affinity constant of 5.4x10(8)/mol.L(-1) and 2.3x10(8)/mol.L(-1), slightly lower than its parental monoclonal antibody E(7)B(10) which had 2.7x10(9)/mol.L(-1).

Construction of Single-chain Antibody to Carcinoembryonic Antigen and Its High Expression in E. coli.

The heavy and light chain variable region (V(H) and V(K)) genes encoding the murine monoclonal antibody E(7)B(10) to carcinoembryonic antigen were linked into single-chain antibody gene with 36 oligonucleotide (Gly(3)Ser)(3) by using recombinant PCR. The construct was highly expressed in E. coli as inclusion bodies (IB), accounting for 20% of the total bacteria proteins, and was characterized by SDS-PAGE and Western blot. When the content of inclusion bodies was denatured and followed by renaturation, it was shown to possess abiliby to kind to its specific antigen CEA by using RIA and competitive ELISA.