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 Table of Contents  
Year : 2020  |  Volume : 11  |  Issue : 3  |  Page : 105-114

Radioimmunotherapy of B-cell lymphoma: In vitro investigations of 177Lu-rituximab on raji cells

Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Submission19-Jun-2020
Date of Decision08-Jul-2020
Date of Acceptance05-Aug-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Dr. Chandan Kumar
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jrcr.jrcr_28_20

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Background: Rituximab is a chimeric monoclonal antibody, approved by the US Food and Drug Administration for the immunotherapy of non-Hodgkin's lymphoma (NHL).177Lu-labeled rituximab has been identified as a potential agent for the radioimmunotherapy of NHL and is presently under clinical investigations. The objective of the present study is to estimate the magnitude of apoptotic cell death and cell-cycle phase arrest. Materials and Methods: Characterization of177Lu-rituximab was performed by using instant thin-layer chromatography as well as by high-performance liquid chromatography. About 37 MBq (1 mCi) of177Lu-rituximab was incubated with Raji cells up to 48 h at 37°C in a humidified atmosphere of 5% CO2. Simultaneously, an equivalent amount of rituximab present in 37 MBq (1 mCi) of177Lu-rituximab complex was used as a vehicle control. All cell samples (treated, vehicle control, and control cells) were harvested post 24 and 48 h of incubation to perform different assays such as lactate dehydrogenase, XTT, cell viability by flowcytometer, apoptosis, and cell cycle analysis. Results: The studies revealed that177Lu-rituximab induced higher cell death and apoptosis compared to unlabeled rituximab. Similarly, an increase in cell population in G1-phase of cell cycle was observed, upon treatment of Raji cells with177Lu-rituximab complex for 24 h, while an increase in G2/M phase population was observed at 48 h of incubation. Conclusions: The present studies demonstrate that177Lu-rituximab is more effective in inducing apoptotic cell death and cell cycle-phase arrest compared to its unlabeled counterpart, indicating177Lu-rituximab may have better potential in the therapy of B-cell lymphoma.

Keywords: 177Lu-rituximab, B-cell lymphoma, cell cycle-phase arrest, non-Hodgkin's lymphoma, radioimmunotherapy

How to cite this article:
Pareri AU, Kambli DB, Amirdhanayagam J, Guleria M, Das T, Kumar C, Dash A. Radioimmunotherapy of B-cell lymphoma: In vitro investigations of 177Lu-rituximab on raji cells. J Radiat Cancer Res 2020;11:105-14

How to cite this URL:
Pareri AU, Kambli DB, Amirdhanayagam J, Guleria M, Das T, Kumar C, Dash A. Radioimmunotherapy of B-cell lymphoma: In vitro investigations of 177Lu-rituximab on raji cells. J Radiat Cancer Res [serial online] 2020 [cited 2021 Nov 30];11:105-14. Available from:

  Introduction Top

Cancer remains as one of the leading causes of death worldwide. The World Health Organization reported that cancer is solely responsible for an estimated 9.6 million deaths in 2018. Blood cancer accounts for 4% of all cancers and is the 7th leading cause of mortality.[1] The National Foundation for Cancer Research, United States (US), reported that one person is diagnosed with blood cancer in approximately every 3 min, and it accounts for almost 10% of new cancer cases in the USA each year. Non-Hodgkin's lymphoma (NHL) is a common hematological malignancy in India.[2] NHL, which affects both the children and adult population, occurs when the B-lymphocytes or T-lymphocytes multiply incessantly and metastasize readily. B-cell lymphomas, which account for >90% of NHL, express a large number of CD20 proteins on the outer surface of the affected cells, and this overexpression of CD20 receptor proteins can be considered a tumor marker for the diagnosis and treatment of patients suffering from NHL.[3],[4],[5],[6],[7] Thus, targeting CD20 plays a crucial role in the treatment of such cancer. This has prompted the development of several anti-CD20 antibodies which are now commercially available for the treatment of such hematological malignancies, thus increasing the choices for providing immunotherapy to the patients suffering from B-cell malignancies.[8],[9],[10],[11]

Immunotherapy, a type of cancer treatment that boosts the body's natural defenses to fight cancer, offers distinct advantages over conventional therapies for cancer. Like chemotherapy, immunotherapy is also a systemic treatment modality, but with a high degree of target specificity and comparatively much lower toxicity.[12] Therefore, immunotherapy holds considerable promise as a modality of cancer treatment, and several immunotherapy drugs have already been approved to fight cancer while hundred more are presently being evaluated clinically. Rituximab is a genetically engineered murine-human monoclonal antibody which specifically binds to the CD20 receptors. It was approved by the US Food and Drug Administration (US-FDA) in 1997 for the treatment of NHL.[13],[14] Rituximab significantly improves treatment outcome in relapsed or refractory, low-grade or follicular B-cell NHL.[15] The expiry of patent, availability of bio-similar antibodies, and absence of human anti-mouse antibody response helped this immunotherapeutic agent to emerge as a choice for the therapy of NHL. However, despite the success of rituximab, patient response is a limiting factor as hardly 50% of the NHL patients showed positive response to rituximab therapy.[16],[17] Such avoidance of therapeutic response is associated with the downregulation of CD20 from the cell surface and overexpressions of various cell survival and anti-apoptotic proteins.[16],[17]

The application of radioisotopes in health care, particularly for the therapy of cancer, has gained considerable momentum in the past two decades. In the recent past,177 Lu has emerged as one of the most preferred radionuclides for radiotherapeutic treatment of cancer patients.[18] Suitable nuclear decay characteristics such as emission of moderate energy β-particles (β[max]= 497 keV), comparatively longer half-life (T½= 6.73 days), and simultaneous emission of imageable gamma photons (208 keV, 11%, and 113 keV, 7%), which allows simultaneous dosimetry studies as well as monitoring the therapeutic course of action makes177 Lu the best choice as a radionuclide for the preparation of therapeutic radiopharmaceuticals.[19],[20] Moreover, possibility of its production through simple neutron irradiation with adequate specific activity and excellent radionuclide purity using medium-flux research reactors have made177 Lu an attractive choice as a radionuclide for developing agents for radiotherapeutic interventions.[21]

The high degree of the target specificity exhibited by the antibodies can also be exploited for the treatment of cancers in the form of radioimmunotherapy (RIT), where a small amount of antibody is labeled with a therapeutic radionuclide to deliver the cytotoxic doses of ionizing radiation to the target cells. RIT offers distinct advantages over immunotherapy, as the cross-fire effect of the radionuclide helps in destroying the cancer cells sitting deep inside the tumor lesions with insignificant receptor expression and where the radiolabeled drug fails to reach through blood circulation.[22],[23] In addition, the internalization process of the antigen–antibody complex begins within an hour after binding to the cells, thus the nucleus gets significant dose of radiation which is sufficient to kill the cancer cells.[14] Clinically, RIT is most widely applied to the most radiosensitive tumors, such as, leukemia and lymphomas and has emerged as a highly promising therapeutic modality with established clinically efficacy, particularly for the treatment of NHL.[15],[23]

The promising potential of the radiolabeled antibodies has already been exploited for the treatment of NHL, and two radiolabeled anti-CD20 antibodies of murine origin, namely Zevalin (labeled with90 Y) and Bexxar (labeled with131 I), have been approved by the US-FDA for clinical use.[22] However, to overcome the drawbacks associated with the use of antibodies of murine origin and to utilize the established potential of177 Lu in targeted radiotherapy, attempts have been directed to develop177 Lu-labeled anti-CD20 antibodies of genetically engineered chimeric and humanized origin. Toward this, several reports have been documented describing the formulation of clinical dose of177 Lu-rituximab and the utilization of such preparations for the treatment of NHL patients.[15],[24] Although various177 Lu-based RIT agents are presently under development and few such preparations are undergoing various phases of clinical investigations for the treatment of B-cell lymphoma, the exact mechanisms by which these agents induce cell death are not well understood, and this is reflected in the limited information available in this subject area in the contemporary literature. Undoubtedly, knowing the cellular and molecular mechanisms of interaction of177 Lu-rituximab with tumor cells and magnitude of induced cell death caused by this agent will play a significant role in understanding the usefulness and efficacy of this agent in the targeted radiotherapy of NHL in a better way. In the present article, an attempt has been made in that direction. Herein we report the detailedin vitro biological evaluation of177 Lu-rituximab in Raji cell line and document the comparative cytotoxic efficacy of the agent over its unlabeled counterpart.

  Materials and Methods Top

Chemicals and reagents

Anti-CD20 antibody rituximab (Biosimilar, rituximab) was obtained in solution (10 mg/mL) form from Dr. Reddy's laboratories, (Hyderabad, India). P- NCS-benzyl-DOTA [2, 2, 2″-(10-(1-carboxy-4-((4 -isothiocyanatobenzyl)amino)-4-oxobutyl)-1, 4, 7, 10-tetraazacyclo-dodecane-1, 4, 7-triyl) triacetic acid] was procured from Macrocyclics, Plano TX (USA).177 LuCl3, used for the present study, was obtained from the Radiochemicals Section of Radiopharmaceuticals Division of our Institute, and produced indigenously following the procedure reported elsewhere.[21] The instant thin-layer chromatography (ITLC) strips were procured from Agilent Technologies CA (USA). The high-performance liquid chromatography (HPLC) studies were carried out using a TSK-Gel G3000SWXL size exclusion column (7.8 mm × 300 mm), which was preequilibrated using phosphate buffer (0.05 M, pH 7.4) maintaining a flow rate of 0.5 mL/min. The elution profile was monitored by following the radioactivity signal using a well-type NaI (Tl) detector coupled with the HPLC system. All the solvents used for HPLC were of HPLC grade and were degassed and filtered prior to use.

Raji (Burkitt lymphoma) cell line was obtained from the Cell Repository of National Center for Cell Sciences, Pune, India. Fetal bovine serum (FBS) and antibiotic/antimycotic solution were obtained from HiMedia Laboratories Pvt Ltd, (Mumbai, India). Roswell Park Memorial Institute 1640 (RPMI-1640), phosphate-buffered saline (PBS), sodium 3′-[1-[(phenylamino)-carbony]-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), sodium dodecyl sulfate, and lactate dehydrogenase (LDH) activity assay kit were obtained from Merck KGaA, Darmstadt, Germany. Guava nexin kit, Guava ViaCount reagent, and Guava cell cycle reagent were purchased from Luminex Corp. Austin, Texas, USA. All radioactive countings were performed using a well-type NaI (Tl) scintillation counter, obtained from Electronic Corporation of India Limited, (Hyderabad, India), by keeping the baseline at 150 keV and using a window of 100 keV so as to utilize the 208 keV gamma photon emission of177 Lu. Color of different assay was determined by ELISA reader BioTek Instrument Inc. VT, USA, and flowcytometer studies were carried out in Guava easyCyte flowcytometer (Luminex, TX, USA) and analyzed by Guava InCyte software from Luminex, TX, USA.

Preparation of177 Lu-rituximab and characterization

Several attempts have been made to radiolabel rituximab with177 Lu and use the preparation as a RIT agent.[25],[26] As177 Lu cannot be directly incorporated into the rituximab moiety, radiolabeling was carried out by using a bifunctional chelating agent (BFCA), namely P-NCS-benzyl-DOTA, and 2 mg of rituximab antibody.[26] The conjugation reaction between the antibody and BFCA was carried out by incubating the mixture of rituximab and p-NCS-benzyl-DOTA in 0.2 M Na2 CO3:NaHCO3 buffer (pH = 9.5) in a molar ratio of 1:10 at 37°C for 17 h. Subsequently, the unlabeled BFCA was removed from the reaction mixture using a prepacked PD-10 column using sodium acetate buffer (pH = 5.5) as the eluting solvent. The concentration of the purified p-NCS-benzyl-DOTA-rituximab was determined by following the standard Bio-Rad protein assay. For the preparation of177 Lu-labeled rituximab, the p-NCS-benzyl-DOTA-rituximab conjugate was incubated with177 LuCl3(100 μL, 1.48 GBq, 40 mCi) in 0.2 M sodium acetate buffer (pH = 5.6) at 37°C for 1½ h. Specific activity of177 LuCl3 was 20 mCi/μg. The percentage radiochemical yield (% RCY) of177 Lu-rituximab was determined by ITLC as well as by HPLC. For ITLC, 0.1 M sodium citrate buffer (pH = 5.0) was used as mobile phase, whereas HPLC chromatogram was recorded in an isocratic mode using 0.05 M sodium phosphate buffer (pH = 6.8) containing 0.05% NaN3 as mobile phase.

Cell culture

Raji cells were grown in RPMI-1640 media supplemented with 10% FBS and antibiotic solution (10 ml/L of 100 × solution). Cells were incubated at 37°C in humidified 5% CO2 atmosphere of an incubator and passaged at every alternate day.

Cell binding of177 Lu-rituximab

In vitro cell binding studies were carried out in Raji cells which are known to overexpress the CD20 receptors. The cell-binding studies were carried out previously to determine the immunoreactivity fraction of177 Lu-rituximab.[26] However, to confirm the specific binding of177 Lu-rituximab toward the CD20 antigen, different concentrations of cells (1 × 106 to 4 × 106) were added in a 24-well plate and a fixed amount of177 Lu-rituximab was added with the cells. The plate was then incubated at 4°C for 2 h. Subsequently, the cells were harvested and washed thrice with 1% FBS containing PBS and centrifuged at 1000 × g for 5 min. The radioactivity associated with cells was measured in well-type NaI (TI) scintillation counter, and percent cell binding was calculated from these data.

Treatment of Raji cells with177 Lu-rituximab

Raji cells (1 × 106) were plated in 6-well microplates containing 5 mL of RPMI-1640 media in each well and incubated for 2 h in the CO2 incubator. Subsequently, the cells were treated with 37 MBq (1 mCi) of177 Lu-rituximab and were kept for 48 h at 37°C in a humidified atmosphere of the incubator. Untreated cells were maintained as the control group, and cells treated with unlabeled-rituximab (equivalent amount of177 Lu-rituximab ~50 μg/mCi) were maintained as the vehicle control. Post 24 and 48 h of the incubation period, the cells were harvested and used for different assays.

Study of cell toxicity induced by177 Lu-rituximab in Raji cells

Lactate dehydrogenase assay

Raji cells were treated with 3.7 MBq (1 mCi) of177 Lu-rituximab and an equivalent amount of rituximab present in 3.7 MBq of177 Lu-rituximab in complete medium for 24 and 48 h. After completion of the incubation period, the cells were harvested and centrifuged, and the supernatant culture medium was collected to determine the amount of LDH released by the cells. The assay was carried out following the protocol of the kit. In brief, the assay mixture (equal volumes of LDH assay substrate, cofactor, and dye) was added to the 96-well plate containing supernatant culture media in the ratio of 2:1 (v/v). The plate was then covered with an aluminum foil to protect the mixture from light and incubated at room temperature for ½ h. The reaction was terminated by adding 1 N HCl (one-tenth of the total volume of reaction) to each well, and absorbance of the reaction mixture was measured at 490 nm using an ELISA plate reader. The percent LDH release was determined for the cells treated with rituximab and177 Lu-rituximab by multiplying the ratio of the optical densities of the treated sample to that of control sample with 100.

Study of cell proliferation by XTT assay posttreatment with177 Lu-rituximab

For studying the percentage of cell proliferation in Raji cells after treatment with rituximab and177 Lu-rituximab, the XTT assays were carried out at 24 and 48 h posttreatment following the protocol briefly described here. The cells were harvested after brief centrifugation. The supernatant was discarded, and the cell pellet formed was resuspended in complete media. Equivalent of 1 × 104 cells (control, vehicle control, and treatment) were placed in a 96-well plate. 50 μL of XTT reagent mixture (1:50 of XTT and activating reagent) was added to each well, and the plate was placed in the CO2 incubator for 4 h, after covering the plates with an aluminum foil. The absorbance of the reaction mixture was recorded at 490 nm using the ELISA plate reader. The percent cell proliferation was determined for the cells treated with rituximab and177 Lu-rituximab by taking ratio of the optical density of the untreated cells to treated cells multiplied by 100.

Study of cell viability induced by177 Lu-rituximab using flow cytometry

The guava ViaCount assay was used to determine cell viability. The assay was performed according to the ViaCount assay kit. The principle of the assay is based on the differential permeability of two dyes, which helps to distinguish between viable and nonviable (dead/apoptotic) cells and allows for the quantitative assessment of viable and nonviable nucleated cells present in a suspension. The cells were harvested after their respective incubation period by centrifugation for 5 min at 1000 rpm. Cell pellet was washed twice with PBS and resuspended in 1 mL of 1% FBS-containing PBS. Around 20 μL of cells were thoroughly mixed with 380 μL of ViaCount reagent, and the reaction mixture was incubated in dark for 5 min. Subsequently, the sample mixture was loaded onto the Guava easyCyte flowcytometer, which provided the absolute counts associated with the viable and dead cells.

Study of apoptotic cell death induced by177 Lu-rituximab in Raji cells

For studying the apoptosis in Raji cells after treatment with177 Lu-rituximab and rituximab, the cells were harvested after 24 and 48 h of incubation and washed twice with 1% FBS-containing PBS. The pellet was resuspended in 1 mL of 1% FBS-containing PBS. From the cell suspension, 100 μL was pipetted out in the micro-centrifuge tubes, and 100 μL of Guava nexin reagent was added in each tube. The tubes were then covered with aluminum foil and incubated at room temperature for 20 min. Subsequently, the samples were loaded onto Guava easyCyte flowcytometer system and analyzed by Guava InCyte software.

Study of cell cycle arrest induced by177 Lu-rituximab

The cell cycle assay was carried out according to the protocol provided in the Guava cell cycle reagent package insert. To study the cell cycle phase arrest, Raji cells were synchronized in G0 phase by culturing the cells for overnight in RPMI 1640 medium without serum. The cells were then treated with 37 MBq (1 mCi) of177 Lu-rituximab. The vehicle control group was maintained by treating the cells with an equivalent amount of rituximab present in the 37 MBq (1 mCi) of177 Lu-rituximab, and untreated cells were considered the control group. The plates were subsequently incubated for 24 and 48 h at 37°C with 5% CO2 in culture conditions. All the cells were maintained in complete media during treatment. The cells were harvested after completion of the designated incubation period and washed twice with PBS. The cells were then fixed by adding 5 mL of 70% ethanol and incubated at 4°C for about 4 h. Subsequently, the cells were washed twice with PBS and resuspended in PBS. 100 μL of the cells were mixed with 100 μL of cell cycle reagent and incubated for ½ h before loading onto the Guava easyCyte flowcytometer system for analysis of the cell cycle phases.

Statistical analysis

All the experiments were carried out in triplicates, and data were analyzed by t-test using “P” value to be <0.05. All the data were represented as mean ± standard deviation.

  Results Top

Radiolabeling and characterization of177 Lu-rituximab

The radiochemical purity of177 Lu-rituximab was determined by ITLC as well as HPLC studies and found to be >95% under the optimized conditions. In ITLC, uncomplexed177 Lu moved with the solvent front (Rf= 0.8–1.0), whereas177 Lu-rituximab remained at the point of spotting (Rf= 0.0–0.1). On the other hand, in HPLC,177 Lu-rituximab eluted from the column at retention time (Rt) 15.5 min, whereas free177 LuCl3 eluted at 21 min. Typical ITLC and HPLC patterns of177 Lu-rituximab, prepared under optimized conditions, are shown in [Figure 1]a and [Figure 1]b, respectively. Specific activity of177 Lu-rituximab was 1.92 mCi/nmol.
Figure 1: (a) Typical instant thin.layer chromatography pattern of177Lu-rituximab. (b) Typical high-performance liquid chromatography pattern of177Lu-rituximab

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Determination of percent cell binding of177 Lu-rituximab

Cell-binding studies were performed using177 Lu-labeled rituximab in Raji cells at 4°C, revealed a cell binding of approximately 28.65% ± 1.52%, 23.31% ± 2.38%, and 21.38% ± 1.2% when 4 × 106, 2 × 106, and 1 × 106 cells were used for the studies, respectively. The results of percentage cell binding of177 Lu-rituximab observed in Raji cell lines with respect to different concentrations of cells at 4°C are shown in [Figure 2]. There was no significant difference in cell binding when cell binding of 4 × 106 cells with 2 × 106 cells was compared; however, a significant difference between cell binding was observed between 4 × 106 cells and 1 × 106 cells at P < 0.05, using t-test.
Figure 2: Binding of177Lu-rituximab with Raji cells at 4°C after 2 h of incubation

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Estimation of cell toxicity in Raji cell using lactate dehydrogenase assay

The LDH is an intracellular enzyme which gets released from the cells which have a damaged cell membrane, and hence is used as a marker for cell toxicity. Raji cells were treated with 37 MBq (1 mCi) of177 Lu-rituximab and were assessed for the release of LDH from the cells after incubation periods of 24 and 48 h. The results of the cell toxicity with respect to percentage LDH released from the affected cells after 24 and 48 h are shown in [Figure 3]. The figure depicts the comparative cellular toxicity exhibited by the Raji cells when treated with rituximab and177 Lu-rituximab separately for 24 and 48 h. It is evident from this figure that the cells treated with177 Lu-rituximab showed statistically, significantly (P < 0.05, t-test) higher extent of cell toxicity compared to those treated with unlabeled rituximab. While an increase of 1–1.2 fold in LDH release was observed in the cells treated with unlabeled rituximab in comparison with the untreated cells, LDH release exhibited a ~2.5 fold increase when the cells were treated with radiolabeled rituximab at 24 h post incubation (P < 0.05, t-test). The cell toxicity was further enhanced when the period of incubation was extended to 48 h as the release of LDH by the cells treated with177 Lu-rituximab showed an almost 5-fold increase in comparison with the control cells (P < 0.001, t-test). This study clearly demonstrates that177 Lu-rituximab has an additional therapeutic effect compared to unlabeled rituximab and causes higher cell cytotoxicity in Raji cells.
Figure 3: Percentage of lactate dehydrogenase released by Raji cells after the treatment of unlabeled rituximab and177Lu-rituximab for 24 h and 48 h

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Study of cell proliferation by XTT assay

For investigating the therapeutic effect of177 Lu-rituximab on Raji cells, percentage decrease of cell proliferation was recorded after treating the cells with unlabeled rituximab (vehicle control) and177 Lu-rituximab for 24 and 48 h incubation. [Figure 4] depicts the results of this experiment, and it clearly shows that the cells treated with177 Lu-rituximab exhibited significantly lesser cell proliferation compared to the cells treated with unlabeled rituximab both after 24 as well as 48 h of incubation time. While the cells treated with177 Lu-rituximab exhibited around 60%–70% decrease in cell proliferation after 24 h of incubation, the cells treated with unlabeled rituximab exhibited a decrease of only 27%–37% under identical conditions (P < 0.001, t-test). Although the difference in retardation of cell proliferation between the cells treated with unlabeled rituximab and177 Lu-rituximab reduced at 48 h Post incubation, the efficacy exhibited by177 Lu-rituximab in decreasing the cell proliferation remained superior compared to that exhibited by unlabeled rituximab (P < 0.05, t-test). This study also indicated that177 Lu-rituximab has superior therapeutic efficacy on Raji cells compared to its unlabeled counterpart.
Figure 4: Percent cell proliferation exhibited by the Raji cells when incubated with unlabeled rituximab and177Lu-rituximab for 24 and 48 h

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Study of177 Lu-rituximab-induced cell viability in Raji cell lines by flow cytometry method

To study the cell viability in Raji cells after treatment with177 Lu-rituximab, the cell samples were analyzed using the Guava ViaCount module. [Figure 5]a and [Figure 5]b depicts the results obtained after treating the Raji cells with 37MBq (1 mCi) of177 Lu-rituximab for 24 and 48 h. The live cells appear on the left side and the dead cells appear on the right side of the plot. It is evident from the figure that177 Lu-rituximab reduces the viability in Raji cells to a greater extent in 48 h compared to that obtained with the cells treated with unlabeled rituximab. Treatment of the cells with177 Lu-rituximab for 24 h reduced the cell viability by 15%–16%, while the reduction of cell viability was only 6%–7% when the cells were treated with unlabeled rituximab under identical conditions (P < 0.05, t-test). When the treatment was continued for 48 h, the cells treated with177 Lu-rituximab showed a significantly higher rate of decrease in viability (52%–53%) compared to the cells treated with unlabeled rituximab (2%–3%) (P < 0.001, t-test).
Figure 5: (a) Cell viability observed in Raji cells treated with177Lu-rituximab (A) control cells, (B) vehicle control cells, and (C) treated cells after 24 h; (D) control cells, (E) vehicle control cells, and (F) treated cells after 48 h. (b) Percentage viability of Raji cells after treatment with unlabeled rituximab and177Lu-rituximab for 24 and 48 h estimated by flowcytometer

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Estimation of apoptotic cell death induced by177 Lu-rituximab

In order to study the mode of cell death induced by177 Lu-rituximab treatment in Raji cells, the magnitude of apoptosis was determined by the Guava Nexin Kit. The result of the assay was obtained by acquiring the samples on the Guava easyCyte flowcytometer system using the Guava InCyte software, and the result of this study is shown in [Figure 6]. The data was displayed in an Annexin V-PE versus 7-AAD dot plot with quadrant markers, which could be adjusted for immediate on-screen results for Annexin V-PE-positive and 7-AAD-positive cells. It was observed that the cells treated with177 Lu-rituximab for 24 h exhibited 28%–30% apoptosis, whereas the cells treated with unlabeled rituximab exhibited 19%–21% apoptosis [Figure 6], (P < 0.05, t-test). On the other hand, the cells treated with177 Lu-rituximab for 48 h showed 82%–88% apoptosis, whereas the cells treated with unlabeled rituximab showed only 32%–38% apoptosis [Figure 6], (P < 0.001, t-test). This study also demonstrates that177 Lu-labeled rituximab has a better efficacy in decreasing the number of viable B-cell compared to unlabeled rituximab.
Figure 6: Apoptosis observed in Raji cells after treatment with unlabeled rituximab and177Lu-rituximab (a) control cells, (b) vehicle control cells, and (c) treated cells after 24 h of incubation; (d) control cells, (e) vehicle control cells, and (f) treated cells after 48 h of incubation

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Analysis of cell-cycle arrest induced by177 Lu-rituximab

Because cancer cells develop when the normal cell cycle-regulating mechanisms are disrupted, it is necessary to understand the genetic basis of this disruption. On performing the Guava cell cycle reagent assay after incubating the cells with177 Lu-rituximab for 24 and 48 h, the software directly provides the information regarding the percentage of cells present in each cell cycle phase depending on the intensity of fluorescence detected in different phases due to the propidium iodide-labeled cellular DNA. The results of cell cycle arrest assay carried out in Raji cells are tabulated in [Table 1]. It is evident from this data that the cells treated with both unlabeled rituximab and177 Lu-rituximab arrested the cells at the G0/G1 phase of the cell cycle after 24 h of incubation (P < 0.05, ANOVA), whereas the arrest of G2/M phase became prominent after 48 h of incubation (P < 0.05, ANOVA).
Table 1: Cell cycle-phase analysis after Raji cells treated with rituximab and177Lu-rituximab for 24 and 48 h

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  Discussion Top

Rituximab is a genetically engineered antibody specific to the CD20 receptors and has been approved by the US-FDA in 1997 for use in immunotherapy of NHL.[6],[8],[9] It is a 144 kDa chimeric mouse/human monoclonal antibody consisting of a glycosylated IgG1 kappa immunoglobin of murine light and heavy chain variable regions (Fab region), and human kappa and gamma-1 constant regions (Fc region).[9],[11] Although rituximab has been effective in the treatment of various types of B-cell lymphomas, including NHL, several cases of acute respiratory reactions such as cough, rhinitis, bronchospasm, dyspnea, and sinusitis have been associated with its use in patients during initial administration.[27] In some cases, NHL patients treated with rituximab-containing chemotherapy developed interstitial pneumonitis and diffused alveolar haemorrhage.[27],[28] As large amounts of antibody are required in immunotherapy and with the emergence of rituximab-induced resistance in immunotherapy, efforts had been directed to enhance the therapeutic efficacy of rituximab.[16],[17],[29] Toward this, Attempts were made to conjugate the antibody with toxins, drugs, and radioisotopes to achieve a better treatment response.[30]

RIT of CD20-positive lymphoma has mainly been studied by using Iodine-131 (131 I)- and Yttrium-90 (90 Y)-labeled antibodies.[31] The use of beta radiations emitted from radiopharmaceuticals is more effective in treating selected cancers. The emission of medium-energy beta particles is sufficient to deposit a radiation dose at millimeter range in a vicinity of tumor cell on which the immunoconjugate is targeted, thus it will be effective for tumor micrometastasis.[23]177 Lu was chosen for the present study as it is one of the potential therapeutic radionuclides which are being used in RIT due to its emission of imageable gamma photons accompanying principle beta emission, comparatively longer half-life, and easy production logistics, established as an excellent radionuclide for targeting micrometastatic tumors.[18] The use of177 Lu with rituximab is more preferable as its handling is less hazardous than131 I, and its beta component gives a more favorable tumor to nontumor ratio than90 Y.[15]177 Lu conjugated with rituximab increases the cytotoxic nature of the antibody, and at the same time minimizes the amount of antibody needed for treatment. This would highly help in minimizing the side effects attributed by the use of rituximab-containing chemotherapy and rituximab alone.[15],[24]

The potential of177 Lu-rituximab in radiotherapeutic intervention of NHL patients has already been documented in contemporary literature.[15],[24] The formulation methodology of the patient dose of177 Lu-rituximab both by wet-chemistry and by using freeze-dried kit has also been reported.[25] Presently,177 Lu-rituximab is under clinical investigations for determining the maximum tolerated dose (MTD) in the treatment of patients with relapsed follicular, mantle cell, or other indolent lymphomas.[15],[32] However, limited studies have been conducted to understand the mechanisms by which177 Lu-ritiximab exhibits its therapeutic action against B-cell lymphoma.[32] Knowing the mechanistic pathway through which177 Lu-ritiximab exerts its cytotoxic effects will definitely be helpful in developing better immunotherapeutic strategies as well as in establishing the dosage required for the clinical translations of NHL treatment.

A survey of contemporary literatures indicates that formulation of177 Lu-rituximab has been achieved in different ways by using either 1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) derivatives or diethylenetetraamine pentaacetic acid (DTPA) as the BFCA.[25],[26],[33] For the present work, p-NCS-benzyl-DOTA was chosen as the BFCA as DOTA derivatives are reported to form thermodynamically stable and kinetically inert complexes with177 Lu.[26] The conjugation of p-NCS-benzyl-DOTA with rituximab is possible as it has an activated carbon region (positive charged) to which a nucleophile (such as –NH2 of lysine residue) from the antibody binds via the formation of a thiourea bond. The rituximab-BFCA conjugate was purified and radiolabeled with177 Lu and characterized following the procedure, which was reported earlier from our group.[26] Subsequently, a series of biological studies were performed using the radiolabeled preparation on Raji cells to study the biological behavior and understand the mechanism of action of this agent.

Various studies conducted on Raji cells during the course of present work clearly indicated that the toxicity caused by177 Lu-rituximab is significantly higher compared to unlabeled rituximab owing to the increase of cytotoxic effects in the combination of rituximab and particulates emerging from177 Lu. Rituximab is known to induce cytotoxicity in Raji cells through various cellular signaling pathways.[34],[35] whereas beta radiation emitted by177 Lu has its own mechanism of cell damage. Thus, the extent of cytotoxicity observed in the studies of LDH assay and XTT assay was greater for the radiolabeled rituximab compared to the unlabeled rituximab. The percentage viability in the cells treated with unlabeled rituximab was higher than the cells treated with177 Lu-rituximab, also indicating the increase of Raji cell toxicity when177 Lu-rituximab was used over unlabeled rituximab as the cytotoxic drug. These studies clearly demonstrate that combination of radiotherapeutic effect of177 Lu with the cytotoxic effect of rituximab certainly enhances the efficacy of the agent.

To further understand the effects of177 Lu-rituximab, apoptotic study and cell cycle assay were carried out with Raji cells. The results showed that177 Lu-rituximab-treated Raji cells arrested the cells at the G0/G1 phase of the cell cycle at 24 h and at 48 h, it induced G2/M phase arrest. It also exhibited greater percentage of apoptotic cells compared to unlabeled rituximab. These studies also prove the enhanced cytotoxic effect of177 Lu-rituximab and suggest the role of beta radiations emitted from177 Lu in killing tumor cells. These results are akin to the observations documented in the literature in connection with the studies related to induce apoptotic cell deaths exhibited by the131 I-labeled rituximab and177 Lu-labeled rituximab.[14],[32],[36]

  Conclusions Top

In the present study, an attempt has been made to understand the comparative cytotoxic potential of177 Lu-rituximab over unlabeled rituximab. An important conclusion which can be drawn from the present work is that beta radiation emitted from the177 Lu-labeled rituximab is more potent in inducing cell toxicity compared to equivalent amount of unlabeled rituximab. It enhances apoptotic cell death in Raji cells, which is dependent on the time of incubation. Similarly, cell cycle phase arrest switched “ON” from G1 to G2/M phase with the increase in the time of incubation with177 Lu-rituximab. Results of the present study demonstrate the importance of RIT using177 Lu-rituximab over immunotherapy with rituximab for the treatment of NHL patients, though further investigations and clinical reports are warranted to supplement the observations made in the present study.


The authors thank the staff members of the Radiochemical Section of Radiopharmaceuticals Division of Bhabha Atomic Research Centre for providing the177 Lu used in the present study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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