Abstract
OBJECTIVE To explore the biodistribution and anti-tumor activity of 131I labeled rituximab injected intratumorally or intraperitoneally in vivo in nude mice bearing Raji human Burkitt’s lymphoma xenograft s.
METHODS The rituximab and the mouse IgG were labeled with Na131I using the IODO-GEN method. BALB/C nude mice were xenograft ed with 131I-Rituximab or 131I-IgG and killed on the 1st, 3rd, 7th, and 15th day after injection. The tumor/non-tumor ratio (T/NT) and the dose injected in each gram of the tissue (%ID/g) from 12 organs or tissues of interest, e.g. tumor, blood, were calculated. The long and short axes of each tumor were measured by calipers at 2-3-day intervals aft er treatment, and the growth inhibition of the tumor was calculated using the MIRD formula.
RESULTS When comparing intraperitoneal injection (IP) and intratumoral injection (IT) of 131I-IgG, intratumoral injection of 131I-rituximab produced a significantly higher tumor/non-tumor ratio in all tissues and organs of interest on the 1st, 3rd, and 7th day, respectively (P < 0.05). The %ID/g of tumor was 1.4-1.7-fold and 1.5-3.7-fold in the IP and IgG IT groups, respectively, but the %ID/g of non-tumors was significantly lower in the IP group and IgG IT group. Similarly, the tumor growth was greatly inhibited by intratumoral injection of the 131I-rituximab, whereas it was less inhibited by other forms of the treatment (P < 0.05). However 131I-rituximab injected intratumorally inhibited tumor growth in a dose-dependent manner. The inhibition rate was less with a low dose (75 μCi) and greater with a high dose (150 μCi), yet the difference was not significant (P > 0.05).
CONCLUSION Tumors can absorb the highest amount of the radiolabelled antibodies, and the tumor/non-tumor ratios in the group with intratumoral injection of the 131I-rituximab resulted in the optimal anti-tumor activity.
keywords
- Iodine-131
- anti-CD20 monoclonal antibody
- non-Hodgkin’s lymphoma (NHL)
- intratumoral injection
- radioimmunotherapy
Introduction
131I has been used primarily as radionuclide for either radioimmunotherapy or imaging due to its advantageous characteristics of high availability, low cost, and ease of labeling. The CD20 antigen is specifically expressed on 90%-95% of B lymphoma cells[1]. In this experiment, we studied the biodistribution and antitumor activities of Rituximab with 131I labeled CD20 monoclonal antibody injected intratumorally or intraperitoneally in vivo in nude mice bearing Raji human Burkitt’s lymphoma xenografts.
Materials and Methods
Materials
Rituximab chimeric anti-CD20 monoclonal antibody was supplied by Roche Pharma (Schweiz) Ltd, mouse IgG was supplied by SIGMA, Na131I solution was purchased from Chengdu Gaotong Isotope Co., and Raji cell lines (human Burkitt’s lymphoma) was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. The level of CD20 expression was determined using flow cytometry.
Antibody labeling protocols and assay of the label activity
The Rituximab and the mouse IgG were labeled with Na131I using IODO-GEN methods, with the ratio of antibody weight (mg) to 131I radioactivity (mCi) being 1:10. The labeled yield and radiochemical purity were measured using paper chromatography, and the immune competence was measured using the method of excessive antigen binding of tumor.
Grouping of experimental animal
BALB/C nude mice were provided by the Institute of Laboratory Animals of the Southern Medical University. The experimental animal model was established by subcutaneously injecting suspensions of 6-7×106 Raji cells in exponential growth phase into the right buttock of the BALB/C nude mice. The nude mice whose maximum diameter of the tumor ranged from 0.5-1.5 cm were selected for use in subsequent experiments. They were randomized respectively into 4 and 6 groups as shown in Table 1 and Table 2. The set of 4 groups was utilized for the biodistrubution experiment in vivo, and the set of 6 groups for the radioimmunotherapy experiment. The control cells were MCF-7 cells and A549 cells.
Grouping of nude mice bearing tumor.
Grouping and treatment protocols.
The dynamic biodistribution of 131I-labeled Rituximab in vivo in the nude mice
The 131I-Rituximab and 131I-IgG were administrated to the nude mice following the predetermined protocols. The mice were imaged using single photon emission computed tomography (SPECT, or less commonly used SPET) on the 1st, 3rd, 7th and 15th day after injection and then killed to harvest the organs or tissues of interest, including blood, tumor, liver, spleen, kidney, heart, lung, stomach, large intestine, small intestine, contralateral leg muscles, and femur. Radioactivity (cpm) was measured using a calibrator. The special radioactivity of the tissue (cpm/g) and the injected dose per gram of tissue (%ID/g) were calculated according to the following formula:
%ID/g = special radioactivity of tissue (cpm/g)/radioactivity of injected labels (cpm)
Cumulative absorbed dose
The injected dose per gram tissue values of the labeled antibodies in nude mice were used to calculate the cumulative absorbed dose in tumor tissue according to the formula recommended by the MIRD Committee[2]. Curve fitting was performed for each group, and the area under the curve was determined using the integration method, which was added with 0.5 μCi (1% injection volume) to obtain the value of Ah/mk (μCi·h/g). This was then applied to the following formula to calculate the cumulative absorbed dose (Gy):
Dk is the absorbed dose of target organ k (rad). Ah is the cumulative activity of source organs (μCi·h). mk is the weight of target organ k (g). np represents non-penetrating radiation. p represents penetrating radiation. △represents the average energy radiation per cumulative activity (g·rad/μCi·h). φ is the absorption coefficient. rk ←→rh means that the target organs are the same as the source organs. ∑np△npφnp(rk←→rh) +∑pd△pφp(rk← →rh) is the absorbed dose of unit cumulative radioactivity in unit time for a given radionuclide and organ. In our experiment, 131I was 0.421 g·rad/μCi·h.
Observation of therapeutic effects
The nude mice were evaluated for tumor growth before treatment and each day after treatment for 39 days. The long and short axes of the tumors were measured with calipers, and the formula of tumor size (short axis2 × long axis)/2, was applied to calculate the tumor size precisely. The growth inhibition of the tumor was calculated by the following formula:
Growth inhibition rate of tumor (%) = 100−(mean tumor size of the treatment group/mean tumor size of the non-treatment group) × 100
Statistical analysis
Data were performed with one-way analysis of variance (ANOVA) using SPSS 10.0 statistical software. A value of P < 0.05 was considered statistically significant.
Results
The labeling yield, radiochemical purity, and immunoreactivity of the 131I-Rituximab and 131I-IgG
The labeling yield, radiochemical purity, and immunoreactivity of the 131I-Rituximab and 131I-IgG are shown in Table 3.
Labeling parameters of the 131I-Rituximab and 131I-IgG.
The T/NT ratio after injection of the 131I-Rituximab and 131I-IgG
As shown in Table 4, the T/NT ratio in the blood of 131I-Rituximab IT group was 7.4, 12.3, and 2.4, respectively on the 1st, 3rd, and 7th day after injection, which was significantly higher than in the IP group and in the 131I-IgG IT group (P < 0.05). Similarly, the T/NT ratios in the other tissues and organs were high in the 131I-Rituximab IT group. However, there was no significant difference in T/NT ratio between 131I-Rituximab IT and IP groups on the 15th day after injection (P > 0.05).
T/NT ratio of different radiolabeled antibodies in different sites of injection after various days.
The %ID/g after injection of the 131I-Rituximab and 131I-IgG
Intratumoral injection of 131I-Rituximab produced a dramatically higher %ID/g in tumors on the day of each injection in comparison to intraperitoneal injection (1.4-17-fold) or to intratumoral injection of 131I-IgG (1.7-3.7-fold), respectively, whereas the opposite result was noticed in non-tumors, aside from a few instances, i.e. the %ID/g in heart tissue measured on the 15th day after injection, and a lower %ID/g in non-tumors in the 131I-Rituximab IT group. For 131I-IgG treatment protocols, the %ID/g in tumors was higher in the 131I-IgG IT group than that in the 131I-Rituximab IP group on the 1st day after injection (42.2 vs. 5.1, P < 0.05). The difference, as shown in Table 5, declined with prolonged time, and by the 3rd day after injection, there were no differences between the 2 groups (P > 0.05). For the mice receiving intratumoral injection of the 131I and rituximab mixture, the %ID/g in tumors was reduced from 192.4 at the moment of injection to 0.6 at 24 h after injection.
The %ID/g of different radiolabeled antibodies in different sites of injection after various days.
Cumulative absorbed dose
As shown in Table 6, the cumulative absorbed dose in the 131I-Rituximab IT group was higher than that in the other groups at each time point of detection. Specifically, it was 12.6-fold and 2.1-fold in the IP group and in the 131I-IgG IT group on the 3rd day after injection, 6.0-fold and 2.5-fold on the 7th day after injection, 7.0-fold and 1.5-fold on the 15th day after injection, respectively.
Cumulative absorption in tumor of each group on different days after injection.
Radioimmunoimaging
Radioimmunoimaging was performed on the 1st, 3rd, and 7th day after injection. As shown in Fig. 1, the tumors were clearly visible in the 131I-Rituximab IT group, but invisible in IP group throughout the study. For the 131I-IgG IT group, tumors were clearly visible on the 1st day after injection, which, however, became blurred on the 3rd day after injection and indistinguishable on the 7th day after injection. There was no recognized tumor observed in 131I + rituximab IT group even on the 1st day after injection.
Radioimmunoimage of nude mice xenografted with tumor after injection with radiolabeled antibody.
Changes in tumor size as a result of the function of treatment protocols
The changes in tumor size are shown in Table 7. In general, tumors increased in size shortly after the initiation of radioimmunotherapy, irrespective of the treatment protocol used. Specifically, tumors grew at a relatively slow speed in groups 2-5 after the initiation of radioimmunotherapy, as shown in Table 7. The tumors became gradually smaller following the 14th day after injection in groups 3-5. Similar behavior was seen in group 2 during the 10th-24th days after injection, but unfortunately, the tumors started to increase in size following the 26th day after injection in group 2. In contrast, tumors in group 1 and 6 grew rapidly at first, while those in group 1 became gradually smaller upon the 19th day after injection, and those in group 6 grew progressively throughout the experiment. These results suggested that both intratumoral and intraperitoneal injection of the 131I-Rituximab had certain inhibitive effects on the growth of Raji lymphoma, however, the former was superior to the latter in therapeutic effects. The IT and IP injection of the 131I-Rituximab had no inhibitive effects on MCF7 or A549 cell tumors, and although intratumoral injection of 131I-IgG inhibited the growth of Raji lymphoma to a some extent, the effects were transient.
The average volume of the tumor in each group on different days after injection.
Comparison of inhibition rate of tumor
Both groups of 131I-Rituximab and 131I-IgG, injected intratumorally or intraperitoneally, exhibited a greater inhibition rate of tumor growth than the control group (P < 0.05). Intraperitoneal injection of the 131I-Rituximab resulted in a lower tumor inhibition rate than the intratumoral injection of 131I-Rituximab. This difference was statistically insignificant when compared with IT 75 μCi group (P = 0.616), but statistically significant when compared with IT 150 μCi group (P = 0.047). The inhibition rate of the tumor was higher in the 131I-Rituximab IT 150 μCi group than in the 131I-Rituximab IT 75 μCi group, but the difference was not significant (P = 0.134). For the 131I-IgG IT group, the inhibition rate of the tumor was lower in the 131I-IgG IT group than in the 131I-Rituximab IT group. When compared with the 131I-Rituximab IT 75 μCi group, the difference was statistically insignificant (P = 0.463) but was statistically significant when compared with the IT 150 μCi group (P = 0.012). The detailed results are presented in Table 8.
The inhibition rate of tumor growth in each group after injection (%).
Discussion
Currently, one inherent obstacle of radioimmunotherapy, suggested by many clinical results, is that radioimmunotherapy administered intravenously, does not generate a high therapeutic concentration of the radiolabelled antibodies in tumors[3-6]. One efficient approach used to circumvent this problem is intratumoral injection of the labeled monoclonal antibody[7-9]. In this experiment, we studied the distribution and antitumor activity of 131I labeled Rituximab injected intratumorally or intraperitoneally in vivo in nude mice bearing Raji human Burkitt’s lymphoma xenografts, which provided some experimental evidence for the clinical applications of radioimmunotherapy.
CD20 is expressed on approximately 90% of Raji cells. 131I-Rituximab, once injected intratumorally, can bind specific CD20 antigens, resulting in great regional radioactivity. In our experiment, the SPECT imaging results showed that the tumor %ID/g, and the cumulative absorbed dose of the radiolabelled antibodies in tumors were much more favorable in the 131I-Rituximab IT group than in the 131I-IgG IT group, which suggested that immunological binding, instead of retention of McAb in tumors, played a role in binding the 131I-Rituximab with the tumor cells.
Similar experiments done previously on tumor-bearing mice often used intravenous or intraperitoneal routes of injection, which generally resulted in a similar biodistribution of the radiolabelled antibodies. Current challenges include[3-6] the labeled antibody being diluted by blood or neutralized by the free antigens in blood circulation, insufficient tumor penetration and nonspecific binding to other tissues. To overcome the above-mentioned unfavorable factors, intratumoral injection was employed in our experiment. Compared with intraperitoneal injection, intratumoral injection reduced the necessity of systematic exposure to the labeled antibody, while the labeled antibodies being bound, whose special radioactivity and immunoreactivity reached the peak, was optimal, resulting in the strongest cytotoxic effects.
In our experiment, the highest %ID/g in tumors was noticed at the moment of injection, and this was followed by a rapid decrease within 72 h and subsequently by a slow reduction. It was evident that binding the anti-CD20 monoclonal antibody to tumor cells was achieved in a short time. The cumulative absorbed dose of the radiolabelled antibodies in tumors in the IT group was 6.0- to 12.6-fold of that in the IP group, yet compared with intraperitoneal injection, intratumoral injection reduced %ID/g lower in non-tumors at each time point. Therefore intratumoral injection led to a favorable therapeutic effect and meanwhile reduced the toxicity and side effects induced by the nonspecific distribution of the radiolabeled antibodies.
The β-emitting 131I is well known for its cytotoxic effects. When the mixture of 131I and anti-CD20 monoclonal antibody was injected intratumorally, 131I was unable to specifically target tumor tissue so that, in our experiment, the %ID/g in tumors was reduced from 192.4 at the moment of injection to 0.6 at 24 h after injection. The culmulative absorbed dose was not enough to kill tumors, which demonstrated that the labeling process of radionuclide played a key role in radioimmunotherapy.
The dose-rate effect of the radioimmunotherapy is generally accepted in clinical practice[10]. Although the relationship among dose-rate effect, tumor repair, and tumor growth rate is complex and beyond the scope of our present experiment, it calls for further studies, as suggested by our experimental results. The 131I-rituximab injected intratumorally inhibited tumor growth in a dose-dependent manner, and the inhibition rate was less with low dose (75 μCi) injection but greater with high dose (150 μCi), whereas the difference was not significant (P > 0.05).
Although the 131I-IgG injected intratumorally had certain inhibitive effects on the growth of Raji lymphoma within a short period, the inhibition rate was reduced to a very low level on the 24th day after injection, indicating that the nonspecific polyclonal antibody was not as effective as the specific monoclonal antibody in terms of tumor growth inhibition. This has been confirmed by many other experimental results done previously[11-15]. Because the CD20 expression on breast cancer MCF7 cells and lung cancer CD20 cells were negative, 131I-Rituximab injected intratumorally had no inhibitive effects on the growth of MCF7 and A549 cell xenografts. Therefore monoclonal antibodies play an important role in radioimmunotherapy.
To conclude, intratumoral injection of the 131I-Rituximab, as our experimental results suggested, resulted in the maximum uptake of the radiolabelled antibodies in tumors and in optimal antitumor activity. Due to the nature of systemic disease, lymphoma should be primarily treated using systematic therapy. To this point, our experimental results have built up a primary foundation for further exploring lymphoma treatment using systematic and regional radioimmunotherapy.
- Received June 8, 2009.
- Accepted July 28, 2009.
- Copyright © 2009 by Tianjin Medical University Cancer Institute & Hospital and Springer
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