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Preliminary evaluation of the potential of 99mTc carbonyl–DTPA–Rituximab as a tracer for sentinel lymph node detection
Applied Radiation and Isotopes 107 (2016) 195–198
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Technical note
Preliminary evaluation of the potential of 99mTc carbonyl–DTPA–Rituximab as a tracer for sentinel lymph node detection Mythili Kameswaran n, Suresh Subramanian, Usha Pandey, Grace Samuel Isotope Production and Applications Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
H I G H L I G H T S
Proposal of 99mTc-labeled Rituximab as a sentinel lymph node (SLN) tracer. Tc carbonyl DTPA–Rituximab obtained in good radiochemical purity. In vitro cell binding studies confirmed presence of higher CD20 receptor density on malignant cells. Tested in Wistar rat using footpad model (popliteal node uptake). Significant SLN uptake and retention of 99mTc carbonyl labeled Rituximab in animal model.
99m
art ic l e i nf o
a b s t r a c t
Article history: Received 17 June 2015 Received in revised form 6 October 2015 Accepted 22 October 2015 Available online 23 October 2015
Preliminary work with 99mTc carbonyl–DTPA–Rituximab was attempted to test its feasibility as a sentinel lymph node (SLN) tracer for patients with breast cancer. 99mTc labeling of DTPA–Rituximab conjugate was carried out via 99mTc carbonyl synthon which exhibited 495% radiochemical purity and good in vitro stability. In vitro studies of 99mTc carbonyl–DTPA–Rituximab in normal and malignant B cells showed higher binding in malignant cells. In vivo distribution of 99mTc carbonyl–DTPA–Rituximab in Wistar rat footpad model indicated good retention by B-cells present in the sentinel lymph node. & 2015 Published by Elsevier Ltd.
Keywords: Rituximab Breast lymphoma Sentinel lymph node Technetium radiopharmaceuticals
1. Introduction In the staging of cancers associated with a well-understood surrounding lymphatic network such as melanoma, breast carcinoma, head and neck cancers etc., Sentinel Lymph Node (SLN) mapping has proved to be a standard care tool that provides useful prognostic information while minimizing the burden of large-scale axillary lymphendectomy on the patient and the clinician alike (Morton et al., 2014; Alkureishi, et al., 2010; Gortzak-Uzan et al., 2010; Veronesi et al., 2010). The basic principle of SLN detection is that the tracer molecule travels from the site of injection adjacent to the primary tumor to the lymph node that directly receives lymphatic drainage from the tumor, i.e. the sentinel node. Once the node is detected, either visually (blue dye) or by use of a radiocolloid tracer, it is excised and sent for biopsy to confirm the presence/absence of malignant cells from the primary tumor, which is an indicator of possible metastasis to secondary regions n
Corresponding author. E-mail address: [email protected] (M. Kameswaran).
http://dx.doi.org/10.1016/j.apradiso.2015.10.025 0969-8043/& 2015 Published by Elsevier Ltd.
(Morton and Chan, 2000). An ideal radiopharmaceutical for SLN detection should show good clearance from the site of injection, sufficient uptake in the sentinel lymph node and minimal activity in secondary nodes and other non-specific regions. It should allow for scintigraphic imaging prior to surgery for preliminary localization and subsequent precise in situ detection using a hand-held gamma probe. 99m Tc-labeled sulfur colloid and human serum albumin (HSA) nanocolloid are the prominent clinically employed radiotracers for SLN detection (Wilhelm et al., 1999; Eshima et al., 2000). These conventional nanoparticulate tracers are retained upon phagocytosis by the macrophages in the lymph node. Particle size is the major criterion for the uptake and retention of these SLN tracers and their optimal size range is a compromise between the rate of clearance from the site of injection and specific retention of tracer in the SLN without undue further passage up the lymphatic channel. To alleviate the dependence on particle size and the associated compromise, research has been directed towards development of receptor-targeted SLN tracers that may be expected to have smaller size and faster kinetics of clearance from the site of injection than the conventional tracers. In recent times, different
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groups including ours have evaluated tracers bearing mannose groups with the intention of targeting the mannose receptors on the macrophages (Vera et al., 2001; Jeong et al., 2004; Tsotakos et al., 2010; Morais et al., 2011; Subramanian et al., 2014). Primary breast lymphoma, accounting for about 0.4–0.5% of all breast malignancies, are CD20 þ B-cell lymphomas with diffuse large B-cell lymphoma (DLBCL) being the most common histologic type (Joks et al., 2011). There is no clear consensus for therapy although chemotherapy alone or in combination with other treatments seems to be the most common choice. Immunotherapy with Rituximab has also been used for B-cell lymphomas in combination with chemotherapy or radiation therapy. Prognosis and effectiveness of individual treatments are generally extrapolated from therapy for other extranodal lymphomas. Recent reports have stated that sentinel node biopsy might influence treatment decisions in clinically node-negative patients with PBL (Jennings et al., 2007). Detection of metastatic involvement of lymph nodes is important for management and prognostic evaluation in breast cancer patients. Rituximab, a chimeric monoclonal antibody, known to bind with CD20 molecule, is administered for therapy of non-Hodgkins' lymphoma and some auto-immune disorders (Cartron et al., 2007). Lymph nodes being a region of differentiation for immune cells are a major site for B-cells and there have been reports of using anti-CD20 antibodies to target B-cells in the lymph node as a means of specific uptake and retention in the node (Fan et al., 2013). In the current work, we have prepared and characterized 99m Tc-labeled Rituximab complex. In vitro cell studies were carried out to confirm the specificity of the radioconjugate to CD20 antigen and in vivo studies to determine its efficacy towards SLN detection in Wistar Rat using the well-established footpad model.
2. Materials and methods Rituximab (MabTheras-10 mg/mL) was procured from Roche Inc., Basel, Switzerland. An in-house 99Mo/99mTc generator was used for eluting pertechnetate (99mTcO4-) for radiolabeling. IsoLink kit vials for preparation of the 99mTc carbonyl synthon were provided free of cost by M/s. Mallinckrodt-Covidien, Holland. The bifunctional chelating agent para isothiocyanato benzyl diethylene triamine penta acetic acid (p-NCS-Bn-DTPA) was procured from M/ s. Macrocyclics (Dallas, TX, USA). All other chemicals and reagents were of analytical grade. Hi-SepTM LSM medium (Hi-Media, India) was used for the separation of lymphocytes from whole blood. Raji cells (Burkitt's lymphoma), expressing CD20 antigens on their surface, was procured from the National Centre for Cell Science (NCCS), Pune, India. AMICON Ultra centrifugal filter devices (MW cut off 10 kDa) and PD-10 Sephadex G-25 columns were procured from M/s. Millipore, India and M/s. GE Healthcare, USA, respectively. Well type NaI (Tl) detector was used for carrying out the in vitro radioactivity measurements. Size exclusion HPLC (SE-HPLC) analyses of the radiolabeled antibody was performed on a JASCO HPLC system (M/s. JASCO, Japan) coupled to a PU 1575 UV/visible detector (M/s. JASCO, Japan) and a NaI (Tl) radioactivity detector (Raytest, Germany) using a TSK gel column (G3000 SWXL; 30 cm 7.8 mm; 5 mm) from TOSOH Bioscience, USA. Radiochromatograms were analyzed using the GINA STAR software (Version 4, M/s. Raytest GmBH, and Germany). Radioactivity measurements for biodistribution studies were performed on an integral line flat-bed NaI (Tl) Scintillation Detector (Harshaw, UK). All animal experiments were carried out after obtaining approval from the Institutional Animal Ethics Committee.
2.1. Conjugation and radiolabeling of Rituximab Conjugation of Rituximab with p-NCS-Bn-DTPA and radiolabeling with 99mTc via the 99mTc carbonyl synthon was carried out as per the method previously described by our group (Pandey et al., 2014). Briefly, a ten times molar excess of p-NCS-Bn-DTPA ligand was added to the antibody at pH 9.0 and incubated at room temperature (25 °C) for 2 h followed by overnight incubation at 4 °C. Subsequent to removal of unreacted p-NCS-Bn-DTPA from the reaction mixture by molecular filtration, the number of DTPA molecules coupled per antibody molecule was estimated by spectroscopic assay using Cu(II)–arsenazo (III) complex as reported by Brady (Brady et al., 2004). For radiolabeling, initially the 99mTc carbonyl synthon [99mTc (H2O)3(CO)3] þ was prepared by adding 99mTcO4- (∼185 MBq) to the IsoLink kit vial and heating the contents of the vial at 100 °C for 20 min in a water bath. Alternatively, 99mTc carbonyl could also be synthesized conventionally following the reported procedures (Alberto et al., 1998; Schibli et al., 2000). In brief, an aqueous solution containing 5.5 mg of sodium borohydride, 4 mg of sodium carbonate and 15 mg of sodium potassium tartrate in 0.5 mL of double distilled water was purged with carbon monoxide gas for 15–20 min. Subsequently, 1 mL of 99mTcO4- ( 185 MBq) was added and the reaction mixture was heated at 75 °C for 30 min. After cooling to room temperature, pH of the mixture was adjusted to ∼7.0–7.5 using 0.3 mL of a 1:3v/v mixture of 1 M phosphate buffer (pH 7.5): 1 N HCl. The 99mTc carbonyl synthon was characterized by HPLC and then added to 0.5 mg of DTPA–Rituximab conjugate in 0.2 mL of 0.05 M phosphate buffer. Antibody labeling reaction was carried out at 37 °C for one hour and the reaction mixture was purified by passing through a PD-10 column using 0.05 M phosphate buffer as the eluting buffer. The 99mTc-carbonyl– DTPA–Rituximab conjugate was characterized by SE-HPLC on a TSK G3000SWXL gel column by isocratic elution using 0.05 M phosphate buffer containing 0.05% sodium azide (pH 6.8) at a flow rate of 0.6 mL/min. 2.2. In vitro studies in normal B-cells and malignant B-cells In order to demonstrate the specific binding of 99mTc-carbonyl– DTPA–Rituximab to lymphocytes, comparison studies wherein in vitro binding and inhibition of the 99mTc-carbonyl–DTPA–Rituximab in normal lymphocytes and lymphoblastoid cells derived from Burkitt lymphoma (Raji cells) were carried out. Normal lymphocytes were harvested from human whole blood by gradient method for lymphocyte separation using Hi-SepTM LSM medium as per the manufacturer's protocol. Briefly, whole blood was collected into EDTA coated tubes and diluted with an equal volume of phosphate buffered saline (PBS). The diluted blood was slowly layered onto Hi-Sep LSM medium (containing polysucrose and sodium diatrizoate, adjusted to a density of 1.077070.001 g/mL) and centrifuged at 400 g for 30 min. The buff colored layer at the interface of plasma and gradient medium, containing the lymphocytes was carefully aspirated and washed twice with PBS at 250 g. After counting by hemocytometry, the cells were suitably diluted in RPMI 1640 medium and used for the cell binding assay. Raji cells were grown as a suspension culture in RPMI1640 medium with 10% fetal bovine serum (FBS) growth supplement at 37 °C, 5% CO2 atmosphere. They were harvested by centrifugation, counted in Neubauer chamber and diluted suitably in RPMI 1640 medium for the cell binding assay. For the cell binding assay, a total reaction volume of 500 ml per tube was maintained. Cells, both Raji cells and peripheral blood lymphocytes (PBL), were taken at a concentration of 1 106 per
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Fig. 1. SE-HPLC elution pattern of 99mTc carbonyl–DTPA–Rituximab on a TSK gel column by isocratic elution with 0.05 M phosphate buffer þ 0.05% sodium azide (pH 6.8) at 0.6 mL/min. [99mTc carbonyl–DTPA–Rituximab conjugate (Rt ¼14.0 min)].
tube and the experiment was performed in triplicate. 99mTc-carbonyl–DTPA–Rituximab at a concentration of 0.7 nM was added per tube and incubated for 2 h at 4 °C. At the end of the incubation, the cells were washed twice with ice-cold PBS, supernatant aspirated and cell pellet measured for bound 99mTc activity in a wellshaped NaI (Tl). Non specific studies were carried out under identical conditions with addition of 700 nM of cold Rituximab. In vitro uptake of the tracer and inhibition by cold Rituximab was expressed as a percentage of the total content of radioactive tracer added to the reaction.
2.3. In vivo radioactivity distribution studies Studies of in vivo distribution and pharmacokinetics of 99mTc-carbonyl–DTPA–Rituximab in animal model were performed in Wistar Rats (adult female, ∼200 g) based on the previously described footpad model (Subramanian et al., 2014). Briefly, the rats were anesthetized with a mixture of Ketamine:Xylazine (10:1) administered intraperitoneally. Under anesthesia, 50 μl of the 99mTc-carbonyl–DTPA– Rituximab complex (∼0.74 MBq activity, 30 nM) was injected subcutaneously into the footpad of the animal. The area was then massaged gently for 60 s with a gauze pad. Appearance of bleeding on the gauze pad was a criterion for rejection. The animals were then kept in segregated sets for different time periods (n¼ 4 per time point for 60 min and 180 min). At 5 min prior to the end of incubation, 50 μl of Patent Blue dye solution (1% w/v in saline) was administered to animals of each set in the same region using an identical protocol, to assist in visual identification of nodes. At the end of the respective incubation periods, they were sacrificed by exposure to carbon dioxide atmosphere. Blood was collected by cardiac puncture and the relevant organs/tissues, including lymph nodes were excised. In vivo radioactivity distribution measurements were carried out on an integral line flat-bed NaI (Tl) scintillation detector. In the footpad model, the popliteal node serves as the sentinel lymph node. Passage of the radiotracer from the footpad injection site into the popliteal node and further on into non-specific regions is determined as a measure of its suitability for use in SLN detection. Activity retained in each organ/ tissue was expressed as a percentage value of the total injected dose (% ID). Popliteal extraction (PE) was calculated as a measure of specific retention of the tracer in the sentinel lymph node using the formula given below (Vera et al., 2001): PE = (% IDpopliteal - % IDiliac) / % IDpopliteal Comparison was drawn with the commercially available HSA nanocolloid based SLN detection kit Nanocolls for assessing the in vivo efficacy of the tested preparation (Subramanian et al., 2015).
3. Results and discussion The number of DTPA molecules per molecule of Rituximab in the DTPA–Rituximab conjugate (prepared using a 1:10 Molar ratio
Fig. 2. In vitro cell binding studies with 99mTc carbonyl–DTPA–Rituximab in Raji cells and peripheral blood lymphocytes (PBL).
Table 1 In vivo radioactivity distribution pattern of 99mTc carbonyl–DTPA–Rituximab in Wistar rats following footpad injection protocol (n¼ 4). Organ/Tissue
%ID (60 min)
%ID (180 min)
Liver Intestine Stomach Kidney Heart Lungs Spleen Blood Urine/Stools 1st Node 2nd Node Site of Injection %PE
17 0.1 0.3 7 0.1 0.17 0.1 0.3 7 0.02 0.17 0.0 0.2 7 0.01 0.0 7 0.0 3.9 7 0.2 0.17 0.1 2.67 0.6 0.47 0.0 75.87 3.9 82.47 4.2
2.2 7 0.5 0.7 7 0.1 0.17 0.1 0.8 7 0.1 0.17 0.1 0.3 7 0.2 0.17 0.1 8.6 7 2.0 2.5 7 1.6 1.3 7 0.1 0.3 7 0.0 66.37 2.4 74.3 7 0.6
of antibody to DTPA) were determined to be five, as per the method described earlier by our group (Pandey et al., 2014). The radiochemical purity of 99mTc carbonyl–DTPA–Rituximab as determined by SE-HPLC was 495% as shown in Fig. 1. In the optimized SE-HPLC system, 99mTc carbonyl–DTPA Rituximab showed a retention time of 14.0 min while free 99mTc carbonyl had a retention time of 22.0 min. In vitro stability studied up to 24 h post preparation showed that the product retained 490% radiochemical purity indicating the stability of the radioimmunoconjugate. In vitro cell binding studies with 99mTc carbonyl–DTPA–Rituximab in Raji cells showed a specific binding of 20.070.23% while studies in PBL showed binding of 11.970.42% indicating the presence of more CD20 receptors on the surface of malignant cells as compared
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to normal cells. Addition of 1000 fold (∼700 nM) excess cold Rituximab showed inhibition of ∼72% and 45% in Raji cells and PBL respectively as shown in Fig. 2 indicating the specificity of the radioconjugate. The results of the in vivo distribution studies are summarized in Table 1. At 60 min p.i. there was a significant uptake of 99mTc carbonyl–DTPA–Rituximab in the popliteal node (2.670.6%ID). The popliteal extraction factor was also quite favorable (82.474.2%), indicating good retention of the radiolabeled Rituximab by the B-cells present in the lymph node in this period. Apart from popliteal and iliac nodes, negligible activity was found further up the lymphatic channel or in other organs. Some amount of tracer was associated with blood (3.970.2%ID), but at 0.370.01% ID/g concentration it is not expected to provide a significant challenge for SLN detection by scintigraphy or in situ. The amount administered activity that remained at the site of injection was 75.572.8%. At 180 min p.i., activity at the site of injection was reduced to 66.3 72.4%ID. However, radioactivity retained in the SLN also decreased (1.370.1%ID) with increase in blood levels (8.6 72.0% ID, 0.5 70.1%ID/g). This could be due to continual degradation and loss of the tracer antibody from the SLN on account of immune system processes after binding with CD20 receptors. But PE factor remained appreciable at 74.3 70.6% and no activity was observed further up the lymphatic channel. When compared with Nanocolls, the in vivo results for 99mTc-carbonyl–DTPA–Rituximab appear less favorable. Following an identical experimental protocol, we found at 180 min better uptake of Nanocolls in the SLN (4.97 1.3%ID) with comparable values for PE (76.4 75.7%) and radioactivity at the site of injection (65.4 7 4.6%ID).
4. Conclusion This preliminary work carried out with 99mTc carbonyl–DTPA– Rituximab was primarily aimed at providing evidence for its use as a sentinel lymph node tracer for patients with breast cancer. Rituximab was successfully labeled with 99mTc using the carbonyl core and tested in Wistar rat footpad model for its suitability as a sentinel lymph node detection tracer. The results of this preliminary study indicate its potential as a SLN tracer for detecting sentinel lymph node (SLN) although further work may need to be carried out to maximize retention of activity in the SLN.
Disclosure statement Research at the Bhabha Atomic Research Centre is part of the ongoing activities of the Department of Atomic Energy, India and is fully supported by government funding.
Acknowledgements The authors acknowledge Dr. A. Dash, Head, Isotope Production and Applications Division for his support during these studies.
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Alkureishi, L.W., Ross, G.L., Shoaib, T., Soutar, D.S., Robertson, A.G., Thompson, R., Hunter, K.D., Sorensen, J.A., Thomsen, J., Krogdahl, A., Alvarez, J., Barbier, L., Santamaria, J., Poli, T., Sesenna, E., Kovács, A.F., Grünwald, F., Barzan, L., Sulfaro, S., Alberti, F., 2010. Sentinel node biopsy in head and neck squamous cell cancer: 5-year follow-up of a european multicenter trial. Ann. Surg. Oncol. 17, 2459–2464. Brady, E.D., Chong, H., Milenic, D.E., Brechbiel, M.W., 2004. Development of a spectroscopic assay for bifunctional ligand–protein conjugates based on copper. Nucl. Med. Biol. 31, 795–802. Cartron, G., Blasco, H., Paintaud, G., Watier, H., Le Guellec, C., 2007. Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit. Rev. Oncol. Hematol. 62, 43–52. Eshima, D., Fauconnier, T., Eshima, L., Thornback, J.R., 2000. Radiopharmaceuticals for lymphoscintigraphy: including dosimetry and radiation considerations. Semin. Nucl. Med. 30, 25–32. Fan, Z., Li, J., Wang, T., Xie, Y., Fan, T., Lin, B., Ouyang, T., 2013. Level III axillary lymph nodes involvement in node positive breast cancer received neoadjuvant chemotherapy. The Breast 22, 1161–1165. Gortzak-Uzan, L., Jimenez, W., Nofech-Mozes, S., Ismiil, N., Khalifa, M.A., Dubé, V., 2010. Sentinel lymph node biopsy vs. pelvic lymphadenectomy in early stage cervical cancer: is it time to change the gold standard? Gynecol. Oncol. 116, 28–32. Jennings, W.C., Baker, R.S., Murray, S.S., Howard, C.A., Parker, D.E., Peabody, L.F., Vice, H.M., Sheehan, W.W., Broughan, T.A., 2007. Primary breast lymphoma: the role of mastectomy and the importance of lymph node status. Ann. Surg. 245, 784–789. Jeong, J.M., Hong, M.K., Kim, Y.J., Lee, J., Kang, J.H., Lee, D.S., Chung, J.K., Lee, M.C., 2004. Development of 99mTc-mannosyl human serum albumin (99mTc-MSA) as a novel receptor binding agent for sentinel lymph node imaging. Nucl. Med. Commun. 25, 1211–1217. Joks, M., Myśliwiec, K., Lewandowski, K., 2011. Primary breast lymphoma – a review of the literature and report of three cases. Arch. Med. Sci. 1, 27–33. Morais, M., Subramanian, S., Pandey, U., Samuel, G., Venkatesh, M., Martins, M., Periera, S.M.S., Correia, J.D.G., Santos, I.R., 2011. Mannosylated dextran derivatives labeled with fac-[M(CO)3] þ (M¼ 99mTc, Re) for specific targeting of sentinel lymph node. Mol. Pharm. 8, 609–620. Morton, D.L., Chan, A.D., 2000. The concept of sentinel lymph node localization: how it started. Semin. Nucl. Med. 30, 4–10. Morton, D.L., Thompson, J.F., Cochran, A.J., Mozzillo, N., Nieweg, O.E., Roses, D.F., Hoekstra, H.J., Karakousis, C.P., Puleo, C.A., Coventry, B.J., Kashani-Sabet, M., Smithers, B.M., Paul, E., Kraybill, W.G., McKinnon, J.G., Wang, H.J., Elashoff, R., Faries, M.B., 2014. Final trial report of sentinel-node biopsy versus nodal observation in melanoma. N. Engl. J. Med. 370, 599–609. Pandey, U., Kameswaran, M., Sarma, H.D., Samuel, G., 2014. 99mTc carbonyl DTPA– Rituximab: preparation and preliminary bioevaluation. Appl. Radiat. Isot. 86, 52–56. Schibli, R., La Bella, R., Alberto, R., Garcia-Garayoa, E., Ortner, K., Abram, U., Schubiger, P.A., 2000. Influence of the denticity of ligand systems on the in vitro and in vivo behavior of 99mTc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules. Bioconjug. Chem. 11, 345–351. Subramanian, S., Pandey, U., Morais, M., Correia, J.D., Santos, I., Samuel, G., 2014. Comparative biological evaluation of two [99mTc(CO)3]-dextran pyrazolyl mannose conjugates developed for use in sentinel lymph node detection. Q. J. Nucl. Med. Mol. Imaging 58, 216–223. Subramanian, S., Pandey, U., Shah, S., Rangarajan, V., Samuel, G., 2015. An indigenous single-vial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection. Nucl. Med. Commun. 36, 848–853. Tsotakos, T., Morais, M., Correiac, J.D.G., Santos, I., Pelecanou, M., Papadopoulos, Pirmettis, I., 2010. New 99mTc(CO)3 mannosylated dextrans bearing S-derivatized cysteine chelator. Nucl. Med. Biol. 37, 680. Vera, D.R., Wallace, A.M., Hoh, C.K., Mattrey, R.F., 2001. A synthetic macromolecule for sentinel node detection: 99mTc-DTPA-Mannosyl-Dextran. J. Nucl. Med. 42, 951–959. Veronesi, U., Viale, G., Paganelli, G., Zurrida, S., Luini, A., Galimberti, V., Veronesi, P., Intra, M., Maisonneuve, P., Zucca, F., Gatti, G., Mazzarol, G., De Cicco, C., Vezzoli, D., 2010. Sentinel lymph node biopsy in breast cancer: Ten-year tesults of a tandomized controlled study. Ann. Surg. 251, 595–600. Wilhelm, A.J., Mijnhout, G.S., Franssen, E.J.F., 1999. Radiopharmaceuticals in sentinel lymph-node detection – an overview. Eur. J. Nucl. Med. 26, S36–S42.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Technical note
Preliminary evaluation of the potential of 99mTc carbonyl–DTPA–Rituximab as a tracer for sentinel lymph node detection Mythili Kameswaran n, Suresh Subramanian, Usha Pandey, Grace Samuel Isotope Production and Applications Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
H I G H L I G H T S
Proposal of 99mTc-labeled Rituximab as a sentinel lymph node (SLN) tracer. Tc carbonyl DTPA–Rituximab obtained in good radiochemical purity. In vitro cell binding studies confirmed presence of higher CD20 receptor density on malignant cells. Tested in Wistar rat using footpad model (popliteal node uptake). Significant SLN uptake and retention of 99mTc carbonyl labeled Rituximab in animal model.
99m
art ic l e i nf o
a b s t r a c t
Article history: Received 17 June 2015 Received in revised form 6 October 2015 Accepted 22 October 2015 Available online 23 October 2015
Preliminary work with 99mTc carbonyl–DTPA–Rituximab was attempted to test its feasibility as a sentinel lymph node (SLN) tracer for patients with breast cancer. 99mTc labeling of DTPA–Rituximab conjugate was carried out via 99mTc carbonyl synthon which exhibited 495% radiochemical purity and good in vitro stability. In vitro studies of 99mTc carbonyl–DTPA–Rituximab in normal and malignant B cells showed higher binding in malignant cells. In vivo distribution of 99mTc carbonyl–DTPA–Rituximab in Wistar rat footpad model indicated good retention by B-cells present in the sentinel lymph node. & 2015 Published by Elsevier Ltd.
Keywords: Rituximab Breast lymphoma Sentinel lymph node Technetium radiopharmaceuticals
1. Introduction In the staging of cancers associated with a well-understood surrounding lymphatic network such as melanoma, breast carcinoma, head and neck cancers etc., Sentinel Lymph Node (SLN) mapping has proved to be a standard care tool that provides useful prognostic information while minimizing the burden of large-scale axillary lymphendectomy on the patient and the clinician alike (Morton et al., 2014; Alkureishi, et al., 2010; Gortzak-Uzan et al., 2010; Veronesi et al., 2010). The basic principle of SLN detection is that the tracer molecule travels from the site of injection adjacent to the primary tumor to the lymph node that directly receives lymphatic drainage from the tumor, i.e. the sentinel node. Once the node is detected, either visually (blue dye) or by use of a radiocolloid tracer, it is excised and sent for biopsy to confirm the presence/absence of malignant cells from the primary tumor, which is an indicator of possible metastasis to secondary regions n
Corresponding author. E-mail address: [email protected] (M. Kameswaran).
http://dx.doi.org/10.1016/j.apradiso.2015.10.025 0969-8043/& 2015 Published by Elsevier Ltd.
(Morton and Chan, 2000). An ideal radiopharmaceutical for SLN detection should show good clearance from the site of injection, sufficient uptake in the sentinel lymph node and minimal activity in secondary nodes and other non-specific regions. It should allow for scintigraphic imaging prior to surgery for preliminary localization and subsequent precise in situ detection using a hand-held gamma probe. 99m Tc-labeled sulfur colloid and human serum albumin (HSA) nanocolloid are the prominent clinically employed radiotracers for SLN detection (Wilhelm et al., 1999; Eshima et al., 2000). These conventional nanoparticulate tracers are retained upon phagocytosis by the macrophages in the lymph node. Particle size is the major criterion for the uptake and retention of these SLN tracers and their optimal size range is a compromise between the rate of clearance from the site of injection and specific retention of tracer in the SLN without undue further passage up the lymphatic channel. To alleviate the dependence on particle size and the associated compromise, research has been directed towards development of receptor-targeted SLN tracers that may be expected to have smaller size and faster kinetics of clearance from the site of injection than the conventional tracers. In recent times, different
196
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groups including ours have evaluated tracers bearing mannose groups with the intention of targeting the mannose receptors on the macrophages (Vera et al., 2001; Jeong et al., 2004; Tsotakos et al., 2010; Morais et al., 2011; Subramanian et al., 2014). Primary breast lymphoma, accounting for about 0.4–0.5% of all breast malignancies, are CD20 þ B-cell lymphomas with diffuse large B-cell lymphoma (DLBCL) being the most common histologic type (Joks et al., 2011). There is no clear consensus for therapy although chemotherapy alone or in combination with other treatments seems to be the most common choice. Immunotherapy with Rituximab has also been used for B-cell lymphomas in combination with chemotherapy or radiation therapy. Prognosis and effectiveness of individual treatments are generally extrapolated from therapy for other extranodal lymphomas. Recent reports have stated that sentinel node biopsy might influence treatment decisions in clinically node-negative patients with PBL (Jennings et al., 2007). Detection of metastatic involvement of lymph nodes is important for management and prognostic evaluation in breast cancer patients. Rituximab, a chimeric monoclonal antibody, known to bind with CD20 molecule, is administered for therapy of non-Hodgkins' lymphoma and some auto-immune disorders (Cartron et al., 2007). Lymph nodes being a region of differentiation for immune cells are a major site for B-cells and there have been reports of using anti-CD20 antibodies to target B-cells in the lymph node as a means of specific uptake and retention in the node (Fan et al., 2013). In the current work, we have prepared and characterized 99m Tc-labeled Rituximab complex. In vitro cell studies were carried out to confirm the specificity of the radioconjugate to CD20 antigen and in vivo studies to determine its efficacy towards SLN detection in Wistar Rat using the well-established footpad model.
2. Materials and methods Rituximab (MabTheras-10 mg/mL) was procured from Roche Inc., Basel, Switzerland. An in-house 99Mo/99mTc generator was used for eluting pertechnetate (99mTcO4-) for radiolabeling. IsoLink kit vials for preparation of the 99mTc carbonyl synthon were provided free of cost by M/s. Mallinckrodt-Covidien, Holland. The bifunctional chelating agent para isothiocyanato benzyl diethylene triamine penta acetic acid (p-NCS-Bn-DTPA) was procured from M/ s. Macrocyclics (Dallas, TX, USA). All other chemicals and reagents were of analytical grade. Hi-SepTM LSM medium (Hi-Media, India) was used for the separation of lymphocytes from whole blood. Raji cells (Burkitt's lymphoma), expressing CD20 antigens on their surface, was procured from the National Centre for Cell Science (NCCS), Pune, India. AMICON Ultra centrifugal filter devices (MW cut off 10 kDa) and PD-10 Sephadex G-25 columns were procured from M/s. Millipore, India and M/s. GE Healthcare, USA, respectively. Well type NaI (Tl) detector was used for carrying out the in vitro radioactivity measurements. Size exclusion HPLC (SE-HPLC) analyses of the radiolabeled antibody was performed on a JASCO HPLC system (M/s. JASCO, Japan) coupled to a PU 1575 UV/visible detector (M/s. JASCO, Japan) and a NaI (Tl) radioactivity detector (Raytest, Germany) using a TSK gel column (G3000 SWXL; 30 cm 7.8 mm; 5 mm) from TOSOH Bioscience, USA. Radiochromatograms were analyzed using the GINA STAR software (Version 4, M/s. Raytest GmBH, and Germany). Radioactivity measurements for biodistribution studies were performed on an integral line flat-bed NaI (Tl) Scintillation Detector (Harshaw, UK). All animal experiments were carried out after obtaining approval from the Institutional Animal Ethics Committee.
2.1. Conjugation and radiolabeling of Rituximab Conjugation of Rituximab with p-NCS-Bn-DTPA and radiolabeling with 99mTc via the 99mTc carbonyl synthon was carried out as per the method previously described by our group (Pandey et al., 2014). Briefly, a ten times molar excess of p-NCS-Bn-DTPA ligand was added to the antibody at pH 9.0 and incubated at room temperature (25 °C) for 2 h followed by overnight incubation at 4 °C. Subsequent to removal of unreacted p-NCS-Bn-DTPA from the reaction mixture by molecular filtration, the number of DTPA molecules coupled per antibody molecule was estimated by spectroscopic assay using Cu(II)–arsenazo (III) complex as reported by Brady (Brady et al., 2004). For radiolabeling, initially the 99mTc carbonyl synthon [99mTc (H2O)3(CO)3] þ was prepared by adding 99mTcO4- (∼185 MBq) to the IsoLink kit vial and heating the contents of the vial at 100 °C for 20 min in a water bath. Alternatively, 99mTc carbonyl could also be synthesized conventionally following the reported procedures (Alberto et al., 1998; Schibli et al., 2000). In brief, an aqueous solution containing 5.5 mg of sodium borohydride, 4 mg of sodium carbonate and 15 mg of sodium potassium tartrate in 0.5 mL of double distilled water was purged with carbon monoxide gas for 15–20 min. Subsequently, 1 mL of 99mTcO4- ( 185 MBq) was added and the reaction mixture was heated at 75 °C for 30 min. After cooling to room temperature, pH of the mixture was adjusted to ∼7.0–7.5 using 0.3 mL of a 1:3v/v mixture of 1 M phosphate buffer (pH 7.5): 1 N HCl. The 99mTc carbonyl synthon was characterized by HPLC and then added to 0.5 mg of DTPA–Rituximab conjugate in 0.2 mL of 0.05 M phosphate buffer. Antibody labeling reaction was carried out at 37 °C for one hour and the reaction mixture was purified by passing through a PD-10 column using 0.05 M phosphate buffer as the eluting buffer. The 99mTc-carbonyl– DTPA–Rituximab conjugate was characterized by SE-HPLC on a TSK G3000SWXL gel column by isocratic elution using 0.05 M phosphate buffer containing 0.05% sodium azide (pH 6.8) at a flow rate of 0.6 mL/min. 2.2. In vitro studies in normal B-cells and malignant B-cells In order to demonstrate the specific binding of 99mTc-carbonyl– DTPA–Rituximab to lymphocytes, comparison studies wherein in vitro binding and inhibition of the 99mTc-carbonyl–DTPA–Rituximab in normal lymphocytes and lymphoblastoid cells derived from Burkitt lymphoma (Raji cells) were carried out. Normal lymphocytes were harvested from human whole blood by gradient method for lymphocyte separation using Hi-SepTM LSM medium as per the manufacturer's protocol. Briefly, whole blood was collected into EDTA coated tubes and diluted with an equal volume of phosphate buffered saline (PBS). The diluted blood was slowly layered onto Hi-Sep LSM medium (containing polysucrose and sodium diatrizoate, adjusted to a density of 1.077070.001 g/mL) and centrifuged at 400 g for 30 min. The buff colored layer at the interface of plasma and gradient medium, containing the lymphocytes was carefully aspirated and washed twice with PBS at 250 g. After counting by hemocytometry, the cells were suitably diluted in RPMI 1640 medium and used for the cell binding assay. Raji cells were grown as a suspension culture in RPMI1640 medium with 10% fetal bovine serum (FBS) growth supplement at 37 °C, 5% CO2 atmosphere. They were harvested by centrifugation, counted in Neubauer chamber and diluted suitably in RPMI 1640 medium for the cell binding assay. For the cell binding assay, a total reaction volume of 500 ml per tube was maintained. Cells, both Raji cells and peripheral blood lymphocytes (PBL), were taken at a concentration of 1 106 per
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Fig. 1. SE-HPLC elution pattern of 99mTc carbonyl–DTPA–Rituximab on a TSK gel column by isocratic elution with 0.05 M phosphate buffer þ 0.05% sodium azide (pH 6.8) at 0.6 mL/min. [99mTc carbonyl–DTPA–Rituximab conjugate (Rt ¼14.0 min)].
tube and the experiment was performed in triplicate. 99mTc-carbonyl–DTPA–Rituximab at a concentration of 0.7 nM was added per tube and incubated for 2 h at 4 °C. At the end of the incubation, the cells were washed twice with ice-cold PBS, supernatant aspirated and cell pellet measured for bound 99mTc activity in a wellshaped NaI (Tl). Non specific studies were carried out under identical conditions with addition of 700 nM of cold Rituximab. In vitro uptake of the tracer and inhibition by cold Rituximab was expressed as a percentage of the total content of radioactive tracer added to the reaction.
2.3. In vivo radioactivity distribution studies Studies of in vivo distribution and pharmacokinetics of 99mTc-carbonyl–DTPA–Rituximab in animal model were performed in Wistar Rats (adult female, ∼200 g) based on the previously described footpad model (Subramanian et al., 2014). Briefly, the rats were anesthetized with a mixture of Ketamine:Xylazine (10:1) administered intraperitoneally. Under anesthesia, 50 μl of the 99mTc-carbonyl–DTPA– Rituximab complex (∼0.74 MBq activity, 30 nM) was injected subcutaneously into the footpad of the animal. The area was then massaged gently for 60 s with a gauze pad. Appearance of bleeding on the gauze pad was a criterion for rejection. The animals were then kept in segregated sets for different time periods (n¼ 4 per time point for 60 min and 180 min). At 5 min prior to the end of incubation, 50 μl of Patent Blue dye solution (1% w/v in saline) was administered to animals of each set in the same region using an identical protocol, to assist in visual identification of nodes. At the end of the respective incubation periods, they were sacrificed by exposure to carbon dioxide atmosphere. Blood was collected by cardiac puncture and the relevant organs/tissues, including lymph nodes were excised. In vivo radioactivity distribution measurements were carried out on an integral line flat-bed NaI (Tl) scintillation detector. In the footpad model, the popliteal node serves as the sentinel lymph node. Passage of the radiotracer from the footpad injection site into the popliteal node and further on into non-specific regions is determined as a measure of its suitability for use in SLN detection. Activity retained in each organ/ tissue was expressed as a percentage value of the total injected dose (% ID). Popliteal extraction (PE) was calculated as a measure of specific retention of the tracer in the sentinel lymph node using the formula given below (Vera et al., 2001): PE = (% IDpopliteal - % IDiliac) / % IDpopliteal Comparison was drawn with the commercially available HSA nanocolloid based SLN detection kit Nanocolls for assessing the in vivo efficacy of the tested preparation (Subramanian et al., 2015).
3. Results and discussion The number of DTPA molecules per molecule of Rituximab in the DTPA–Rituximab conjugate (prepared using a 1:10 Molar ratio
Fig. 2. In vitro cell binding studies with 99mTc carbonyl–DTPA–Rituximab in Raji cells and peripheral blood lymphocytes (PBL).
Table 1 In vivo radioactivity distribution pattern of 99mTc carbonyl–DTPA–Rituximab in Wistar rats following footpad injection protocol (n¼ 4). Organ/Tissue
%ID (60 min)
%ID (180 min)
Liver Intestine Stomach Kidney Heart Lungs Spleen Blood Urine/Stools 1st Node 2nd Node Site of Injection %PE
17 0.1 0.3 7 0.1 0.17 0.1 0.3 7 0.02 0.17 0.0 0.2 7 0.01 0.0 7 0.0 3.9 7 0.2 0.17 0.1 2.67 0.6 0.47 0.0 75.87 3.9 82.47 4.2
2.2 7 0.5 0.7 7 0.1 0.17 0.1 0.8 7 0.1 0.17 0.1 0.3 7 0.2 0.17 0.1 8.6 7 2.0 2.5 7 1.6 1.3 7 0.1 0.3 7 0.0 66.37 2.4 74.3 7 0.6
of antibody to DTPA) were determined to be five, as per the method described earlier by our group (Pandey et al., 2014). The radiochemical purity of 99mTc carbonyl–DTPA–Rituximab as determined by SE-HPLC was 495% as shown in Fig. 1. In the optimized SE-HPLC system, 99mTc carbonyl–DTPA Rituximab showed a retention time of 14.0 min while free 99mTc carbonyl had a retention time of 22.0 min. In vitro stability studied up to 24 h post preparation showed that the product retained 490% radiochemical purity indicating the stability of the radioimmunoconjugate. In vitro cell binding studies with 99mTc carbonyl–DTPA–Rituximab in Raji cells showed a specific binding of 20.070.23% while studies in PBL showed binding of 11.970.42% indicating the presence of more CD20 receptors on the surface of malignant cells as compared
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to normal cells. Addition of 1000 fold (∼700 nM) excess cold Rituximab showed inhibition of ∼72% and 45% in Raji cells and PBL respectively as shown in Fig. 2 indicating the specificity of the radioconjugate. The results of the in vivo distribution studies are summarized in Table 1. At 60 min p.i. there was a significant uptake of 99mTc carbonyl–DTPA–Rituximab in the popliteal node (2.670.6%ID). The popliteal extraction factor was also quite favorable (82.474.2%), indicating good retention of the radiolabeled Rituximab by the B-cells present in the lymph node in this period. Apart from popliteal and iliac nodes, negligible activity was found further up the lymphatic channel or in other organs. Some amount of tracer was associated with blood (3.970.2%ID), but at 0.370.01% ID/g concentration it is not expected to provide a significant challenge for SLN detection by scintigraphy or in situ. The amount administered activity that remained at the site of injection was 75.572.8%. At 180 min p.i., activity at the site of injection was reduced to 66.3 72.4%ID. However, radioactivity retained in the SLN also decreased (1.370.1%ID) with increase in blood levels (8.6 72.0% ID, 0.5 70.1%ID/g). This could be due to continual degradation and loss of the tracer antibody from the SLN on account of immune system processes after binding with CD20 receptors. But PE factor remained appreciable at 74.3 70.6% and no activity was observed further up the lymphatic channel. When compared with Nanocolls, the in vivo results for 99mTc-carbonyl–DTPA–Rituximab appear less favorable. Following an identical experimental protocol, we found at 180 min better uptake of Nanocolls in the SLN (4.97 1.3%ID) with comparable values for PE (76.4 75.7%) and radioactivity at the site of injection (65.4 7 4.6%ID).
4. Conclusion This preliminary work carried out with 99mTc carbonyl–DTPA– Rituximab was primarily aimed at providing evidence for its use as a sentinel lymph node tracer for patients with breast cancer. Rituximab was successfully labeled with 99mTc using the carbonyl core and tested in Wistar rat footpad model for its suitability as a sentinel lymph node detection tracer. The results of this preliminary study indicate its potential as a SLN tracer for detecting sentinel lymph node (SLN) although further work may need to be carried out to maximize retention of activity in the SLN.
Disclosure statement Research at the Bhabha Atomic Research Centre is part of the ongoing activities of the Department of Atomic Energy, India and is fully supported by government funding.
Acknowledgements The authors acknowledge Dr. A. Dash, Head, Isotope Production and Applications Division for his support during these studies.
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