Background: Multiple Myeloma (MM) is a malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for the 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) was produced by the digestion of Tocilizumab with papain for 24h 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 72h. 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 72h 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 surgical excision, if necessary.

Introduction: Multiple Myeloma (MM) is a haematological malignancy in which Interleukin-6 (IL-6) plays a crucial role in its growth and spread. The monoclonal antibody Tocilizumab binds strongly to IL-6 receptors, inhibiting their function. Our aim is to evaluate the potential of [177Lu]Lu-labeled Toclilzumab as a theranostic agent for MM. Methods: Tocilizumab was derivatised with DOTA-NHS ester and radiolabeled with [177Lu]Lu. The stability of the radiochemical compound was evaluated, and in vitro binding and immunoreactive fraction assays were performed. Biodistribution was assessed at 24 and 48 h post-injection (n = 5) in normal and MM-bearing BALB/c nude mice. Results: [177Lu]Lu-DOTA-Tocilizumab remained stable in all tested solutions. Epitope recognition was confirmed by cell-binding studies, and an immunoreactive fraction of 83.7% was observed. In vivo biodistribution studies revealed high tumor uptake over time, with tumor-to-muscle ratios of 11.66 ± 3.81 and 9.54 ± 1.75 at 24 and 48 h, respectively. Discussion: This study focused on the synthesis and characterisation of a new radiolabeled theranostic agent against IL6R, [177Lu]Lu-DOTA-Tocilizumab. This agent demonstrated high radiochemical stability and strong binding to IL-6R in the MM cell line. Biodistribution studies indicated selective retention of the agent in tumor tissue, highlighting its potential for targeted imaging and therapy in IL-6R-positive cancers and paving the way for future research into its application in related diseases. Conclusions: [177Lu]Lu-DOTA-Tocilizumab represents a potential theranostic agent for MM. Therefore, we expect this agent to open the way to new diagnostic and therapeutic strategies for this devastating disease.

Background: Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake. Objective: The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging. Methods: Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice. Results: Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies. Conclusions: We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.

World J Nucl Med, Official publication of World Association of Radiopharmaceutical and Molecular Therapy (WARMTH),Singapore