Background: Multiple myeloma (MM) is malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for proliferation and survival of MM cells and the overexpression of IL-6 receptor is being studied as a molecular target for therapeutic and diagnostic use in myelomas and other comorbidities. Tocilizumab is a humanized monoclonal antibody that binds IL-6R. Objective: We aim to label and evaluate Fab(Tocilizumab) with 99mTechnetium or Cy7 as potential MM imaging agents. Methods: IL-6R distribution was analyzed by laser confocal microscopy (LCM) in MM cell lines. Fab(Tocilizumab) were produced by digestion of Tocilizumab with papain for 24 h at 37 °C, derivatized with NHS-HYNIC-Tfa and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and SPECT/CT were performed. Also, Fab(Tocilizumab) was labeled with Cy7 for in vivo fluorescence imaging up to 72 h. Results: LCM analysis demonstrates IL-6R distribution on MM cell lines. Incubation with papain resulted in complete digestion of Tocilizumab and exhibited a good purity and homogeneity. Radiolabeling with 99mTc via NHS-HYNIC-Tfa was found to be fast, easy, reproducible and stable, revealing high radiochemical purity and without interfering with IL-6R recognition. Biodistribution and SPECT/CT studies showed a quick blood clearance and significant kidney and MM engrafted tumor uptake. Cy7-Fab(Tocilizumab) fluorescent imaging allowed MM1S tumor identification up to 72 h p.i. Conclusion: These new molecular imaging agents could potentially be used in the clinical setting for staging and follow up of MM through radioactive whole-body IL-6R expression visualization in vivo. The fluorescent version could be used for tissue sample evaluation and to guide the surgical excision if necessary.

El factor de crecimiento endotelial vascular (VEGF), es un factor clave en la angiogénesis tumoral de muchos tipos de tumores, incluyendo el Linfoma No Hodgkin (LNH). El objetivo del presente trabajo es la radiomarcación de los fragmentos de unión al antígeno (Fab) del 99m anticuerpo monoclonal Bevacizumab con Tc y su evaluación como potencial agente de imagen para LNH. Para lograrlo se analizó la expresión de VEGF mediante citometría de flujo en una línea celular de LNH (Toledo). La fragmentación se realizó empleando papaína y los Fab obtenidos fueron conjugados con NHS-HYNIC-Tfa y 99m radiomarcados con Tc. La pureza radioquímica y la estabilidad fueron ensayadas. Se realizaron estudios de biodistribución tanto en ratones sanos como en portadores de LNH. Se observó que las células Toledo presentaron una elevada expresión de VEGF. El radiomarcaje se realizó de forma rápida, sencilla, reproducible y estable, con purezas radioquímicas >90%. Los estudios de biodistribución revelaron una significativa captación renal y tumoral, indicando que la principal vía de eliminación es renal. De los resultados 9 9 m obtenidos se concluye que el Tc-HYNIC-Fab (Bevacizumab) representa un potencial a agente de imagen de la expresión de VEGF asociada a LNH.

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.

ChiTn, a mouse/human chimeric anti-Tn monoclonal antibody, was radiolabeled with iodine-131 (131I) and technetium-99m (99mTc) to assess its biodistribution and internalization in Tn-expressing (Tn+) and wild-type (Tn-) LL/2 lung cancer cells. Selective accumulation and gradual internalization of ChiTn were observed in Tn + cells. Biodistribution in mice with both Tn + or Tn- lung tumors indicated that the uptake of radiolabeled ChiTn within tumors increased over time. Dual-labeling experiments with 99mTc and 131I showed different biodistribution patterns, with 99mTc exhibiting higher values in the liver, spleen, and kidneys, while 131I showed higher uptake in the thyroid and stomach. However, tumor uptake did not significantly differ between Tn + and Tn- tumors. To improve tumor targeting, Losartan, an antihypertensive drug known to enhance tumor perfusion and drug delivery, was investigated. Biodistribution studies in Losartan-treated mice revealed significantly higher radiolabeled ChiTn uptake in Tn + tumors. No significant changes were observed in the uptake of the control molecule IgG-HYNIC-99mTc. These findings demonstrate the enhanced tumor targeting of radiolabeled ChiTn in Losartan-treated mice with Tn-expressing lung tumors. They highlight the potential of ChiTn as a theranostic agent for cancer treatment and emphasize the importance of Losartan as an adjunctive treatment to improve tumor perfusion and drug delivery.

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