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ORIGINAL ARTICLE
Ahead of print publication  

Fate of 177Lu-CHX-A”-DTPA-Rituximab: In vitro Evaluation in Raji Cell Line


1 Department of Biotechnology, V. G. Vaze College of Arts, Science and Commerce, Mumbai, Maharashtra, India
2 Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Submission25-Feb-2022
Date of Decision22-Apr-2022
Date of Acceptance23-Apr-2022
Date of Web Publication24-Aug-2022

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

DOI: 10.4103/jrcr.jrcr_15_22

  Abstract 

Context: Radioimmunotherapy is an emerging treatment modality for various types of cancers. While immunotherapy using monoclonal antibodies has shown promising results, particularly in hematological malignancies, a significant number of patients develop resistance to the treatment, which may be overcome using monoclonal antibodies labeled with suitable therapeutic radioisotopes. Aim: In this study, in vitro evaluation studies of 177Lu-CHX-A''-DTPA-rituximab were performed in Raji cells that overexpress CD20. The extent of internalization of 177Lu-CHX-A”-DTPA rituximab inside the target cell as well as the impact of cellular toxicity in Raji cells was studied. Materials and Methods: The monoclonal antibody rituximab was labeled with 177Lu using CHX-A”-DTPA as the bifunctional chelator. In vitro cell binding and inhibition studies were performed in Raji cells to ascertain the specificity of the radioimmunoconjugate toward the CD20 receptors. The immunoreactive fraction was determined to evaluate the integrity of the radioimmunoconjugate. A cellular internalization assay was performed to evaluate the extent of internalization of the radioimmunoconjugate, and the extent of cytotoxicity was determined using flow cytometry in comparison with unlabeled rituximab. Results: Radiochemical purity of 177Lu-CHX-A''-DTPA-rituximab was determined to be 97.4% ± 1%. In vitro cell-binding studies in Raji cells showed a cell concentration-dependent increase in the percent cell binding, which surged from 11.7% ± 0.7% to 22.7% ± 0.9%, as the cell concentration increased from 0.94 × 10^6 to 7.5 × 10^6 successively. Inhibition in binding was observed in the presence of unlabeled rituximab (11.7% ± 0.7% to 7.8% ± 1.2% and from 22.7% ± 0.9% to 12.1% ± 1.3%). The immunoreactive fraction was found to be 78.5%. A time-dependent increase in the cellular internalization from 25.21 ± 1.7 to 60.47 ± 0.20 was observed. The percent cell viability decreased from 56% to 41% when the cell was treated with rituximab compared with 177Lu-rituximab. Conclusions: Thus, the results show a potential of 177Lu-rituximab as a promising radiopharmaceutical against non-Hodgkin's lymphoma.

Keywords: Immuno-reactive fraction, non-Hodgkin's lymphoma, radioimmunotherapy, radiopharmaceutical, rituximab



How to cite this URL:
Samant SA, Kumar C, Pandey U. Fate of 177Lu-CHX-A”-DTPA-Rituximab: In vitro Evaluation in Raji Cell Line. J Radiat Cancer Res [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.journalrcr.org/preprintarticle.asp?id=354445


  Introduction Top


Cancer is the second most fatal disease afflicting people all over the world. According to the estimate of the American Cancer Society, in the United States only, around 1,898,160 new cancer cases and 608,570 cancer deaths are projected to occur in the year 2021.[1] Among the various cancers, lymphoma accounts for about half of the total blood cancers occurring every year. Non-Hodgkin's lymphoma (NHL) is ranked as the 5th to 9th most common cancer in most countries worldwide, with almost 510,000 new cases estimated in 2018.[2] Efforts are continuing to develop effective treatment methods for various cancers. In spite of the best efforts, the choice of treatment becomes very difficult due to the unpredictability of irreversibly dividing cells having the capability of invasion leading to metastasis. While chemotherapeutic drugs remain the mainstay of the treatment regimen along with surgery, there are reports of inefficacy of several chemotherapeutic drugs for some cancers due to lack of specificity, high toxicity, poor biodistribution, and damage to the normal tissues.

The past few decades have witnessed significant advancements in the fields of molecular biology, medicine, and radiochemistry and led to the emergence of radioimmunotherapy (RIT) as a treatment modality for targeting several cancers. RIT, in particular, has shown maximum efficacy in the treatment of NHL, as evidenced by preclinical and clinical studies in patients.[3] Two radio-immunotherapeutics, namely Bexxar and Zevalin are Food and Drug Administration (FDA) approved for therapy of NHL.[4] The high specificity of the monoclonal antibodies to the tumor associated antigens directs the radio-immunotherapeutic agent to the tumor.[5] The cytotoxic radiation emitted by the β-emitter enables the killing of cancer cell also by means of cross-fire and bystander effects. In the development of NHL targeting radio-immunotherapeutic, the most important target of interest is the CD20 found on the surface of all normal B-cells and significantly overexpressed on malignant B-cells.[6]

Rituximab is a chimeric monoclonal antibody highly specific to the CD20 antigen, and hence, it is routinely used as an immunotherapeutic agent targeting B-cell NHL.[6] However, treatment cycles with rituximab lead to resistance due to which tumor response is compromised and the cancer relapses in a significant proportion of the patients.[7] The success of RIT using the β-emitter 90Y (Zevalin) has encouraged the evaluation of 177Lu-labeled monoclonal antibodies as RIT agents for several cancers. Advantages of 177Lu include its suitable nuclear decay characteristics as well as the feasibility of production in multi-Curie quantities by thermal neutron activation of 176Lu enriched targets in nuclear reactors.[8] Lutetium-177 decays with a half-life of 6.65 days and emits medium energy β-radiations (Eβmax = 177.0 keV [11.6%], 385.4 keV [9.1%], and 498.3 keV [79.3%]).[8] Emission of low energy gamma radiations Eγ = 112.9 keV (6.2%) and 208.4 keV (10.4%) permits simultaneous imaging.[9] In the design of a therapeutic radiopharmaceutical, the metallic radioisotope is bound to the biomolecule (peptide/monoclonal antibody etc.,) through a bifunctional chelating agent (BFCA). The BFCA functions as a linker between the radioisotope and the biomolecule.[10] p-NCS-Bn-DOTA (para isothiocyanato benzyl 1, 4, 7, 10-tetra azacyclododecane 1, 4, 7, 10-tetra acetic acid) and p-NCS-Bn-CHX-A''-DTPA ([(R)-2-Amino-3-(4-isothiocyanatophenyl) propyl]-trans-[S, S]-cyclohexane-1,2-diamine-pentaacetic acid) are the commonly used BFCAs for labeling of biomolecules such as monoclonal antibodies for RIT and are reported by many research groups including ours.[11],[12],[13],[14],[15],[16] Previously, we had reported on the formulation of patient doses of 177Lu-labeled rituximab employing p-NCS-Bn-CHX-A''-DTPA as the BFCA. The work reported earlier includes the standardization of radiolabeling methodology to obtain stable radioimmunoconjugate, preliminary bio-evaluation studies including in vitro cell binding studies in Raji cells and biodistribution studies.[12],[14]

In this work, extensive in vitro cell studies of 177Lu-CHX-A''-DTPA-rituximab in Raji cells are reported. Raji cell line was chosen for the in vitro cell-binding studies as it overexpresses CD20 on its cell surface, for which rituximab (anti-CD20 antibody) is highly specific. The work performed includes the evaluation of the fate and effectiveness of 177Lu-CHX-A''-DTPA-rituximab in Raji cells by determining the degree of internalization of the radioimmunoconjugate inside the target cell and the amount of cellular toxicity induced by it.


  Materials and Methods Top


Materials and instruments

Rituximab (Reditux®) was purchased from Dr. Reddy's Laboratories Ltd., India. 177LuCl3 is indigenously produced and processed at Radiopharmaceuticals Division, BARC.[17] The specific activity of 177LuCl3 produced was in the order of 20–22 Ci/mg. The ligand p-NCS-Bn-CHX-A''-DTPA was purchased from Macrocyclics (Dallas, TX, USA). Sodium acetate, YCl3.6H2O, and Arsenazo III were purchased from Sigma, USA. AMICON Ultra 4 centrifugal filters (MWCO 10,000 Da) employed for the purification of CHX-A''-DTPA-rituximab conjugate were from Millipore, India. PD-10 columns were obtained from GE Healthcare, USA. Whatman 3 mm paper for radiochemical purity (RCP) determination was procured from Whatman, UK. Size exclusion-high-performance liquid chromatography (SE-HPLC) analyses were done on a TSK gel column (G3000 SWXL) having integrated ultraviolet-visible (UV/VIS) (JASCO) and NaI (Tl) radioactivity detectors (Raytest, Germany). The SE-HPLC chromatograms were analyzed using GINA STAR software (Raytest GmBH, Germany). A Chemito Spectrascan UV2600 Spectrophotometer (M/s. Thermo Scientific) was used for UV absorbance measurements. Radioactive countings were performed using a well-type NaI (Tl) scintillation counter (Raytest, Germany) set for the detection of 177Lu radioactivity. Fetal calf serum (FCS) and antibiotic/antimycotic solution, Roswell Park Memorial Institute 1640 (RPMI-1640), phosphate-buffered saline (PBS), were obtained from Merck Millipore India. Guava ViaCount reagents were purchased from Luminex Corp. Austin, Texas, USA. Flow cytometery studies were performed in Guava easyCyte flow cytometer (Luminex, TX, USA) and analyzed by Guava InCyte software from Luminex, TX, USA.

Cell culture

Raji cell line (CD20 wild type, Burkitt lymphoma) was procured from the National Centre for Cell Sciences, Pune, India. RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) along with 1% antibiotic/antimycotic solution was used to culture the Raji cells. Cells were allowed to grow by incubation at 37°C in a humidified atmosphere of 5% CO2. The cells were passaged after reaching confluence by centrifuging at 1200 rpm for 5 min.

Conjugation of p-NCS-Bn-CHX-A”-DTPA with rituximab

Conjugation of p-NCS-Bn-CHX-A-DTPA with rituximab was carried out following our previous report.[12],[14] Protein concentration in the purified BFCA-antibody conjugate was determined using the method reported by Lowry using IgG as standard.[18] Protein concentration was also estimated by measuring the absorbance of a diluted sample at 280 nm. Spectroscopy assay using yttrium (III)–arsenazo complex was used to determine the number of CHX-A''-DTPA molecules linked per rituximab molecule.[19] The purified immunoconjugate in 0.1 M sodium acetate solution of pH 6–7 was stored at 4°C.

Radiolabeling of rituximab with 177Lu and its characterization

Labeling of p-NCS-Bn-CHX-A''-DTPA-rituximab with 177Lu was carried out following our previous report.[12],[14] 177Lu-CHX-A”-DTPA-rituximab conjugate was characterized by SE-HPLC on a gel column and by paper chromatography (PC). PC using a 10 mM sodium citrate solution (pH = 5) was performed to determine the RCP of the product. The radioimmunoconjugate was also characterized by SE-HPLC using 0.05 M sodium phosphate solution (pH 6.8) containing 0.05% sodium azide as the mobile phase in isocratic mode at a flow rate of 0.6 mL/min for 30 min. Radioactivity and UV absorbance profiles were followed with the aid of integrated radioactivity and UV Vis detectors.

In vitro cell-binding and inhibition assay

In vitro cell-binding and inhibition studies were carried out in Raji cells to establish the specificity of 177Lu-CHX-A''-DTPA-rituximab toward CD20 which is present on the cell surface. The cultured Raji cells were counted using a hemocytometer and different numbers of cells were seeded in 24-well plates.

Cell-binding study

177Lu-CHX-A”-DTPA-rituximab conjugate (~1.5 ng/925 Bq) was added to a range of cell concentrations (0.9 × 106–7.5 × 106) of Raji cells and incubated at 4°C for 2 h. After incubation, the cells were harvested and washed thrice with PBS (containing 1% FCS) by centrifugation at 2000 rpm. Radioactivity associated with cells was counted using a well-type NaI (TI) scintillation counter. The percentage of cell binding was calculated.

Inhibition assay

177Lu-CHX-A”-DTPA-rituximab (~1.5 ng/925 Bq) was added to different concentrations (0.9 × 106–7.5 × 106) of Raji cells just after the addition of unlabeled rituximab (100 times concentration of the radioimmunoconjugate) and incubated at 4°C for 2 h. Cells were then harvested and washed thrice with PBS (containing 1% FBS) by centrifugation at 2000 rpm. Percent inhibition of cell binding was calculated.

Determination of immunoreactive fraction of 177Lu-CHX-A”-DTPA-rituximab

Toward determining the immunoreactive fraction (IRF) of the radioimmunoconjugate, Raji cells were harvested, counted, and seeded in 24-well plates in a range of cell concentrations (0.94 × 106–20 × 106 cells per well). Subsequently, cells were incubated with a fixed amount of the radioimmunoconjugate (~1.5 ng/925 Bq) for 2 h at 4°C. After the incubation period, cells were washed thrice with PBS (containing 1% FBS) and counted in a well-type NaI (Tl) counter. A double inverse plot of cell concentration against cell-bound radioactivity was plotted, and the IRF value was calculated by the equation (100× [1/Y-intercept]) from the graph.[20]

Cell internalization study

Raji cells cultured in flasks were harvested, centrifuged, and counted using hemocytometer. Around 2 × 106 cells were seeded onto 6-well plates. The cells were then exposed to a fixed amount (~1.5 ng/925 Bq) of 177Lu-CHX-A”-DTPA-rituximab, and the plates were incubated for 2 h at 4°C. After completion of the incubation period, the cells were harvested and centrifuged to discard the supernatant. The cell pellet was washed thrice with PBS (containing 1% FBS), and the radioactivity was counted using a gamma counter. The cells were then incubated and radioactivity count was taken at 2 h for the membrane-bound and cellular internalization. To estimate the membrane-bound and internalized radioactivity, cells were incubated with acidic buffer (pH ~ 2.5) for 5 min and centrifuged to separate supernatant and cell pellets. While the radioactivity counts in the cell supernatant correspond to membrane-bound radioactivity, radioactivity associated with cell pellet corresponds to the amount of internalized radioactivity.

Cytotoxicity assay

Raji cells cultured in flasks were harvested and 1 × 106 cells were plated in each well of 6-well plates along with 5 mL of RPMI-1640 medium and incubated at 37°C for 2 h under 5% CO2 atmosphere. Cells were then exposed to 1 mCi (37 MBq) of 177Lu CHX-A''-DTPA-rituximab followed by an incubation period of 48 h. Cells that were not exposed to the radioimmunoconjugate were called controls. Cells that were subjected to treatment with unlabeled rituximab (60 μg) were called vehicle controls. After incubation, the cells were harvested, washed twice with phosphate buffer saline (containing 1% FBS), and 20 μL of the cell suspension was mixed with 380 μL of Guava ViaCount reagent. Reaction mixture was incubated in dark for minimum 5 min at ambient temperature and then loaded onto the flow cytometer for determining the cell viability.[21]

Statistical analysis

All experiments were carried out in triplicates; results were presented as mean ± standard deviation of all the experiments. ANOVA was used to analyze the significant difference between control, vehicle control, and treated samples where P value was considered for <0.05.


  Results Top


Conjugation of p-NCS-Bn-CHX-A”-DTPA with rituximab

Under the optimized reaction conditions, the amino group of rituximab reacted with the isothiocyanate group of p-CHX-A-DTPA-NCS to form a thiourea bond. While the BFCA to antibody molar ratio used for the conjugation was ten to one, four to six molecules of CHX-A-DTPA were conjugated to a molecule of rituximab as evidenced by the results of the spectroscopy assay.

Radiolabeling of rituximab with 177Lu and its characterization

The radiolabeling protocol standardized by our group earlier was followed for 177Lu labeling of CHX-A-DTPA-rituximab.[12],[14] The pure radioimmunoconjugate eluted in the second fraction (out of four fractions of 2.5 mL each) from the PD-10 column, and its RCP was determined both by SE-HPLC and PC. The RCP was determined as 97.4% ± 1%. The specific activity of 177Lu-CHX-A''-DTPA-rituximab was determined to be 0.45–0.65 MBq/μg. RCP of pure radioimmunoconjugate was also determined by PC and it was >95%. In this system, 177Lu-labeled rituximab remained at the origin (Rf = 0), while unbound 177Lu (III) migrated to the solvent front (Rf = 0.8–1.0).

Evaluation of specificity using cell binding and inhibition assay

In vitro cell binding and inhibition assay confirmed the specificity of 177Lu-CHX-A”-DTPA-rituximab toward the CD20 receptor on Raji cells. [Figure 1] depicts an increase in the percentage of cell binding with increase in the cell concentration. The percent cell binding increased from 11.7% ± 0.7% to 22.7% ± 0.9% as the cell concentration increased from 0.94 × 106 to 7.5 × 106. The graph in [Figure 1] also shows decrease in binding of 177Lu-CHX-A”-DTPA-rituximab to the CD20 receptors in the presence of excess amount of unlabeled rituximab. As the concentration of Raji cells increases from 0.94 × 106 cells to 7.5 × 106 cells, the binding of 177Lu-CHX-A”-DTPA-rituximab is reduced from 11.7% ± 0.7% to 7.8% ± 1.2% (33.3% inhibition) and from 22.7% ± 0.9% to 12.1% ± 1.3% (46.7% inhibition) due to the competition from unlabeled rituximab. Thus, the binding of 177Lu-rituximab to the CD20 receptor in the cells is found to decrease in the presence of unlabeled rituximab, confirming the specificity of the radioimmunoconjugate toward CD20 receptor on Raji cells.
Figure 1: Cell-binding and inhibition assay for evaluation of receptor specificity of 177Lu-CHX-A”-DTPA-rituximab in Raji cells (There was a significant difference observed within the same group of binding and inhibition, analyzed using t-test, P < 0.05)

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Determination of immunoreactivity fraction of 177Lu-CHX-A''-DTPA-rituximab

The conjugation of the monoclonal antibody with the BFCA followed by radiolabeling might alter its biological activity due to structural modifications. Hence, it is imperative to determine the structural integrity of the radioimmunoconjugate by means of IRF. IRF was calculated from the graph of inverse of cell concentration versus inverse of cell-bound activity for 177Lu-rituximab [Figure 2]. The IRF of 177Lu-CHX-A''-DTPA-rituximab in Raji cells was calculated from the graph as 78.5% (100* 1/1.2732).
Figure 2: Graph of inverse of cell concentration versus inverse of cell-bound activity of 177Lu-CHX-A”-DTPA-rituximab in Raji cells

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Cell internalization assay

The total percent membrane-bound activity and percent internalized activity of the radioimmunoconjugate in Raji cells (at 2 h) were calculated. It was found that 33.5% ± 0.2% of total bound activity was membrane bound, while 65.5% ± 0.2% of total bound radioactivity is internalized. The development of antitumor radioimmunoconjugates requires the monoclonal antibodies to internalize inside the tumor cells in order to carry out the tumor killing instead of just getting bound to the membrane.[22] The higher percentage of internalization indicates an effective potential of the radioimmunoconjugate to carry out the elimination of tumor cells.

Estimation of cell viability using flow cytometer

The percentage cell viability assay was performed using flow cytometry wherein the ViaCount reagent and Guava ViaCount software was used to determine the cell viability. The percentage of cell viability was found to be 56.3% when the cells were treated with unlabeled rituximab. However, the percentage of viability cells decreased to 41.5% when subjected to treatment with 177Lu-CHX-A''-DTPA-rituximab [Figure 3]. Thus, treatment of Raji cells with 177Lu-labeled rituximab is more effective than the treatment carried out with only rituximab, as 177Lu-rituximab has enhanced cytotoxic effects on the cells due to the β-radiations from 177Lu.
Figure 3: Percent cell viability, apoptosis, and cell death in Raji cells after 48 h of treatment (ANOVA was used to analyze the significant difference between control, vehicle control, and treated samples where P value was considered for <0.05). This figure is representative of three replicates

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  Discussion Top


NHL is a cancer of the lymphatic system and is one of the most common cancers affecting people worldwide. Due to the increasing resistance of tumor cells toward various chemotherapeutic drugs, it is necessary to modify the conventional methods of treatment to increase their efficacy. Inhibitions of cell death mechanism, DNA damage repair, and drug target modification are the most probable mechanisms of drug resistance by cancer cells.[23] RIT using anti-CD20 antibodies has proved to be effective in the treatment of relapsed lymphomas.[24] While FDA-approved radioimunoconjugates targeting NHL are available, with the increased utility of 177Lu-based radiopharmaceuticals, there is significant interest on 177Lu-labeled monoclonal antibodies for therapy.[20],[21] The aim of this work was to perform detailed in vitro studies of 177Lu-CHX-A”-DTPA-rituximab in Raji cells which overexpress CD20. Antibodies are labeled with various radionuclides using different chelators. CHX-A”-DTPA is preferred over other chelators for radiolabeling of heat-labile biomolecules. It can rapidly form thermodynamically stable complexes at room temperature with variety of metal ion with better pharmacokinetics.[25] When antibodies are structurally modified or radiochemically modulated, there is a chance of their effectiveness getting altered in the process, which can be assessed by in vitro evaluation in suitable cell lines. 177Lu-CHX-A”-DTPA-rituximab could be prepared in >95% RCP. The cell-binding and inhibition assay carried out using Raji cells proved the specificity toward the anti-CD20 receptor present in Raji cells. The radiolabeled antibody depicted a high percentage of internalization, thus proving it to be a potent antitumor antibody which agrees with the previous studies with 131-rituximab.[26] The ViaCount Reagent is a mixture of two DNA-binding dyes which distinguishes viable and nonviable cells based on the differential permeabilities. This proprietary dye enables to distinguish between viable, apoptotic, and dead cells. These studies gave an estimate of percent cell viability after treatment with 177Lu-labeled rituximab and unlabeled rituximab. 177Lu-CHX-A”-DTPA-rituximab resulted in an increased cytotoxic effect in Raji cells than the unlabeled rituximab, as the radioimmunotherapeutic agent exerts an additional cytotoxic effect due to the β-radiations which increases the tumor cell death.[27],[28],[29] This study on the determination of fate of 177Lu-CHX-A”-DTPA-rituximab in Raji cell line gives important insights for further development of 177Lu-labeled monoclonal antibodies to augment the success rate in targeting various types of cancers.


  Conclusions Top


Although there are several radiolabel rituximab that are under limited clinical trials for possible use in clinics, 177Lu-labeled rituximab has its own advantage. 177Lu is labeled with a variety of chelating agents such as DOTA and NOTA, but CHX-A”-DTPA is preferred over all since it is labeled at room temperature and avoids damage of antibody secondary structure, and thus, immunoreactivity retains after labeling. Hence, 177Lu-CHX-A”-DTPA-rituximab is considered as potential radioimmunotherapeutic agents for NHL.

Acknowledgment

The first author is grateful to BARC for allowing to conduct the experiments and all the help rendered by the staff members of radiopharmaceuticals division.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

 
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Abstract
Introduction
Materials and Me...
Results
Discussion
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