Cy7-Tocilizumab/Fab(Tocilizumab): Near Infrared Fluorescence In Vivo Imaging of Multiple Myeloma
Authors: Camacho Ximena , Perroni Carolina , de Souza Junqueira Mara , Fernandez Marcelo , Chammas Roger , Buchpiguel Carlos , Gambini Juan Pablo , Cabral Pablo , Riva Eloisa 
Published: October 2019

Abstract
Introduction:
Multiple Myeloma (MM) is an incurable bone marrow cancer characterized by the proliferation of malignant plasma cells. Interleukin-6 (IL-6) is a key player molecule related to growth, survival and proliferation of MM cells. Tocilizumab is a humanized monoclonal antibody directed against the soluble and membrane IL-6 receptor. We have previously radiolabeled Tocilizumab and its Fab´s fragments with 99mTechnetium, revealing its potential role as MM radiotracers for targeting IL-6 expression in vivo [1, 2]. Near-infrared (650-900 nm) fluorescence (NIRF) cyanine (Cy) dye has been used for labeling several molecules for optical imaging due to their properties, such as small size, good aqueous solubility, and pH insensitivity between pH 3 and 10.
Objectives: Label Tocilizumab and Fab´s (Tocilizumab) with Cy7 and evaluate its potential role as MM imaging agent.
Methodology: IL-6R expression in MM cell lines (U266, NCI-H929 and MM1S) was confirmed by laser confocal microscopy. Tocilizumab fragmentation was carried out with papain and, once purified, fragments were identified by MALDI/TOF and SDS-PAGE. Cy7-Tocilizumab / Fab(Tocilizumab) were synthesized through nucleophilic substitution reaction between monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and Tocilizumab / Fab(Tocilizumab) [3]. After purification, the conjugates were characterized by spectrophotometry. For in vivo imaging, Cy7-Tocilizumab/Fab(Tocilizumab) (1 nmol) were injected intravenously in MM1S tumor-bearing Balb/c nude mice and were imaged with near-infrared fluorescence (NIRF) after 0, 1, 2, 6 and 24 h post-injection of Cy7-Tocilizumab and after 0.5, 2, 6, 24, 48 and 72 h post-injection of Cy7-Fab(Tocilizumab).
Results: Laser confocal microscopy showed that MM cell lines express high levels of IL-6R. Pure and homogeneous Fab-Tocilizumab fragments were produced. Tocilizumab and Fab (Tocilizumab) were successfully labeled with Cy7 as shown by spectrophotometry. Non-invasive NIRF in vivo imaging of MM tumor-bearing Balb/c nude mice allowed us to distinguish tumors up to 24 h and 72 h post-injection of Cy7-Tocilizumab and Cy7-Fab(Tocilizumab), respectively (Figures A and B).
Conclusions: MM cell lines express IL-6R. Cy7 labeled Tocilizumab/Fab(Tocilizumab) has the potential to become optical imaging agents for IL-6R expressing tumors such as MM, being useful to guiding surgical excision of tumors and biopsies, that merits further evaluation.
Acknowledgments: Agencia Nacional de Innovación e Investigación - Uruguay (ANII), Roche Laboratories, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay)) and Comisión Sectorial de Investigación Científica-Universidad de la República-Uruguay (CSIC, UdelaR) I+D Grupos Oncología Nuclear.
Disclosures: No relevant conflicts of interest to declare.
References: Camacho, X; Machado, CL; García, MF; Fernández, M; Oddone, N; Benech, J; Gambini, JP; Cerecetto, H; Chammas, R; Cabral, P; Riva, E. "Tocilizumab labeling with 99mTechnetium via HYNIC as a molecular diagnostic agent for multiple myeloma". Anticancer Agents Med Chem, 17(9):1267-1277, 2017. Camacho, X; Machado, CL; García, M; Fernández, M; Alonso, O; Cerecetto, H; Chammas, R; Gambini, JP; Cabral, P; Riva, E. " 99mTechnetium-Tocilizumab fragments as molecular imaging agent for multiple mieloma. Blood. 126:4214, 2015. Camacho, X; Machado, CL; García, MF; Gambini, JP; Banchero, A; Fernández, M; Oddone, N; Bertolini Zanata, D; Rosal, C; Buschpiguel, CA; Chammas, R; Riva, E; Cabral, P. "Technetium-99m- or Cy7-Labeled Rituximab as an Imaging Agent for Non -Hodgkin Lymphoma". Oncology, 15(92):229-42, 2017.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.