99m. Tc-Rituximab as Radiotracer For Molecular Imaging In Non Hodgkin Lymphoma
Authors: Camacho Ximena , Banchero Agustina , García Maria Fernanda , Fernández Marcelo , Oddone Natalia , Moreno María , Guillermo Cecilia , Gambini Juan Pablo , Cabral Pablo , Riva Eloísa 
Published: October 2019

Abstract

Introduction
Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy and the fifth leading cause of cancer death in the world; over 90% are B cell lymphomas and express CD-20 surface antigen [1]. CD-20 antigen is a transmembrane protein of 33-37 kDa protein located on the surface of pre-B and mature lymphocytes B [2, 3]. It is expressed in 90% of B-cell NHL [4]. Anti-CD20 antibody (Rituximab®), a specific chimeric monoclonal antibody directed against CD20 on B lymphocytes and the first monoclonal antibody approved for the treatment of CD20+ NHL. The development of new agents directed against specific targets may improve the sensitivity and specificity of imaging for its staging.

Objective
To radiolabel Rituximab with 99mTc and evaluate its properties as a potential imaging agent for NHL.

Methodology
Rituximab was derivatized with succinimidyl-hydrazinonicotinamide (Suc-HYNIC) and MALDI TOF/TOF was used to confirm the level of HYNIC conjugation to Rituximab. This antibody was radiolabeled with 99mTc using a mixture of Tricine/SnCl2.2H2O. Radiochemical purity was determined by ITLC-SG, size exclusion chromatography and HPLC. In-vitro stability was studied in solution; serum and L-Cysteine up to 24 h. In-vitro binding and competition assays were performed with Ramos and Raji NHL cell lines up to 120 min and were analyzed by laser confocal microscopy. Ramos and Raji cells were incubated with buffer to evaluate any degree of autofluorescence. Biodistribution studies were performed in normal Balb/C mice at 1, 4, 24 and 48 h (n = 5).

Results
HYNIC- Rituximab was efficiently labeled with 99mTc. The in-vitro radiochemical stability studies of the radiolabeled antibody showed that the complexes formed were stable and no significant transchelation was detected. In-vitro binding and displacement assays confirm that after derivatization and labeling, Rituximab retained its specificity of binding to CD-20 antigen.
Immunoreactivity of HYNIC-Rituximab to Ramos and Raji cell lines was determined by direct immunofluorescence. We observed a remarkable cell membrane staining of the NHL cells. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). After its incubation with buffer, auto fluorescence was discarded. These results show that Rituximab´s affinity for NHL cells remained unaffected after its derivatization.
Biodistribution studies were performed to quantify localization and clearance of the radiolabeled antibody complex in normal tissues. Significant accumulation of radioactivity was found in liver, indicating that the primary route of clearance was hepatic. Other major uptake was not found after evaluating the rest of the organs at the observed time points.

Conclusions
99mTc-HYNIC-Tocilizumab was easily and rapidly labeled, with radiochemical purities greater than 90%, retaining its specificity of binding. Our results indicate that 99mTc-HYNIC-Rituximab represents a promising molecular imaging agent for NHL.

Acknowledgments
ANII, Roche Laboratories, Pro.In.Bio. PEDECIBA Química

References
[1] Swerdlow AJ. Epidemiology of Hodgkin’s disease and nonHodgkin’s lymphoma. Eur J Nucl Med Mol Imaging 2003; 30 Suppl 1:S2–12.
[2] Einfield DA, Beown JP, Valentine MA, et al. Molecular cloning of the human cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 1988; 7, 711-717.
[3] Valentine MA, Meier KE, Rossie S, et al. Phosphotylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase. C J Biol Chem 1989; 264, 11282-11287.
[4] Tedder TF, Boyd AW, Freedman As, et al. The B cell surface molecule B1 is functionally linked withBcell activation and differentiation. J Immunol 1985; 135, 973-979.

Disclosures:
No relevant conflicts of interest to declare.