Biological Response of B-Cell Lymphoma to ¹³¹i-Rituximab

Objective:

1. To investigate the technique of labelling ¹³¹I-Rituximab and establish an easy, fast and effective method for iodine-labelled antibodies. To detect the stability and immunoactivity of the labelled compound.

2. To study the biological response of B-cell lymphoma cells to ¹³¹I-Rituximab.

3. To observe the tissue distribution and the therapeutic effect of ¹³¹I-Rituximab in lymphoma tumor borne in nude mice, in order to offer the experimenta ecidence for the next radioimmunotherapy.

Methods:

1. Rituximab was labelled with ¹³¹I by means of Iodogen method. Detecting the labelling efficiency, radiochemical purity and immunoreactive fraction of ¹³¹I-Rituximab at different reaction time. Measuring the changes of radiochemical purity of ¹³¹I-Rituximab at different storage conditions(stores at 4°C).

2. Study the biological character of Raji cells by measuring the cytoactivity by Type Blue, the expression of surface antigens by flow cytometry(FCM), The cells were randomized into four groups that were ¹³¹I-Rituximab group ARituximab alone A¹³¹I alone Ablank group, and its effects on apoptosis and cell cycles of Raji cells were determined by cytometry.

3. Raji cell was inoculated subcutaneously in 35 nude mice. Observe the tissue distribution and the therapeutic effect of ¹³¹I-Rituximab in lymphoma tumor borne in nude mice. Nude mice were randomized into six groups while the tumor was 8 mm in diameter. The groups were: high,middle,low dose radioimmunotherapy groups,Rituximab alone,¹³¹I alone Ablank group. The size of the tumor was measured per three days and the mice were killed in four weeks after treatment. The tumors were resected and weighed.

Results: 1.The labelling efficiency of ¹³¹I and Rituximab at 4 minutes reaction time was 90.0%, and the radiochemical purity was 99.4%, the immunoreacive fraction of ¹³¹I- Rituximab was 35.6%. The compound was satble,after 24h, the radiochemical purity was 91.0%. 2.The Raji cells had more than 90% of cytoactivity and 98.03% of surface expression of CD20. The apoptosis rate measured by Annexin V-FITC/PI was higher than that in other groups. Cell cycle alteration occurred in ¹³¹I-Rituximab group, and the majority of cells were arrested at G² stage. 3.The tissue distribution indicate that ¹³¹I-Rituximab was mainly detected in the tumor. As compared to blank group, therapy groups’ tumor growth was inhibited and the inhibition was does and time dependent. The pathological results show that the therapy is effective.

Conclusion:

1. The labelling method-Iodogen method proved to be effective as the labeling rate was high, the compound was stable, immunoreactive to Raji cells. According to our research, the optimal reaction time for labelling Rituximab with Na¹³¹I is 4 minutes. the labelling efficiency(90.0%), radiochemical purity(98.4%) and immunoactivity were all high very much, and can be obtained for intraurmor injection at animal tests.

2. ¹³¹I-Rituximab can regulate the cycle of Raji cells and induce their apoptosis to inhibit their proliferation.

3. The results indicate that the radioimmunological drug made from anti-CD20 monoclonal antibody and ¹³¹I can specifically localise in tumor tissue and ensure radioimmunological targeting therapy, so it has underlying clinical value.