Introduction: Rituximab was the first monoclonal antibody approved for the treatment of B-cell non-Hodgkin lymphoma (NHL) expressing CD20 antigen. This antibody has also the potential to be used as a specific fluorescent and radiolabel agent for targeting NHL. Objective: To radiolabel rituximab with technetium-99m (^99mTc) or Cy7 and evaluate both probes as potential imaging agents for NHL. Methods: Rituximab was derivatized with the trifluoroacetyl hydrazino protected form of succinimidyl ester of HYNIC and radiolabeled with ^99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) were performed. Raji cells were transfected with luciferase for bioluminescent NHL imaging up to 21 days. Rituximab was labeled with Cy7 for in vivo noninvasive fluorescence imaging up to 96 h. Results: Radiolabeling was carried out in a fast, reproducible, easy, and stable way with high radiochemical purity and did not interfere with epitope recognition. Biodistribution and SPECT/CT studies showed high liver and discrete tumor uptake. Bioluminescence and fluorescence studies helped us evaluate rituximab-Cy7 in Raji subcutaneous engraftment in BALB/c nude mice. Conclusions: Our results support the potential use of rituximab labeled either with ^99mTc or Cy7 as a molecular imaging tool for staging, restaging, and guiding surgical excision of tumors, which merits further evaluation.

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.

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20–30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling β1-integrins into large punctate clusters at the leading edge of tumor cells to promote an “adhesive switch,” decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease.

Our results support the potential role of 99mTc-HYNIC-Tocilizumb as a novel MM radiotracer for targeting IL-6 expression in-vivo. We describe the development of a formulation kit to radiolabeling monoclonal antibodies in a clinical setting. We hope that these novel molecular imaging agents will open the path to new diagnostic and therapeutic strategies for MM disease.

Introduction: Multiple myeloma (MM) is a neoplasm of B lymphoid line that is characterized by clonal proliferation of malignant plasma cells in the bone marrow, producing monoclonal paraprotein (M) in blood and/or serum. Interleukin-6 (IL-6) is one of the key molecules related to growth, survival and proliferation of MM cells. Tocilizumab (TCZ) is a humanized monoclonal antibody directed against IL-6 receptor (IL-6R). When radiolabeled and used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor-to-blood and tumor-to-normal tissue ratios with renal clearance. The aim of our work was to develop a 99mTc radiolabeled TCZ Fab´s fragment and to perform its chemical and biological evaluation in order to be used as a potential MM imaging agent for staging and restaging. Methods: Antibody fragmentation was carried out with papain and, once purified, Fab´s(TCZ) fragments were identified and derivatized with NHS-HYNIC-Tfa as bifunctional coupling agent. MALDITOF/TOF was used to confirm all procedures. A mixture of Tricine/SnCl2.2H2O was added to Fab´s(TCZ)-Tfa-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity and in-vitro stability in saline, serum and different concentration of L-cysteine up to 4 h were analyzed by ITLC and HPLC. In-vitro binding assays were performed using U266 and MM1S cell lines up to 120 min. Biodistribution and SPECT/CT images were evaluated on healthy Balb/c mice and MM1S tumor-bearing Balb/c nude mice at 0.5, 2 and 4 h. Results: Radiolabeling of HYNIC-Tfa-Fab´s(TCZ) was carried out in a fast, reproducible, easy, stable way showing high radiochemical purity and high specific activity. In vitro binding assays confirm that after its derivatization and radiolabeleing, Tfa-HYNIC-Fab`s(TCZ) does not interfere with the epitope recognition. In vivo biodistribution studies on healthy Balb/c mice and MM1S tumor-bearing Balb/c mice showed that 99mTc-HYNIC-Fab´s (TCZ) has significant renal uptake with neglectable uptake in other organs, indicating renal clearance. Tumor uptake was 12.84±1.80 %ID/g followed by 8.94±0.61 %ID/g and 3.05±1.49 %ID/g at 2 and 4 h, respectively. U266 tumor-to-muscle ratios were 5.79, 8.61 and 2.71 at 0.5, 2 and 4 h, respectively.Tumor uptake for MM1S tumor-bearing Balb/c nude mice was 10.05±1.32 %ID/g, 8.59±2.36 %ID/g and 3.88±0.68 %ID/g at 0.5, 2 and 4 h, respectively. MM1S tumor-to-muscle ratios were 6.32, 4.61 and 3.08 at 0.5, 2 and 4 h, respectively. Biodistribution data of 99mTc-HYNIC-Fab´s(TCZ) on U266 tumor-bearing Balb/c nude mice showed good tumor uptake and retention 0.5 h after its injection SPECT/CT images on healthy Balb/c mice and MM1S tumor-bearing Bal/c nude mice of 99mTc-HYNIC-Fab´s(TCZ) showed renal uptake and a discrete tumor uptake at 4 h p.i (Figure 1). Conclusions: Labeling Fab´s(TCZ) with 99mTc using HYNIC was performed in an easy, fast, stable and reproducible way preserving its biological activity. Biodistribution and SPECT/CT imaging assays allowed us to observe and evaluate its potential role as a diagnostic molecular imaging agent for MM. Disclosures No relevant conflicts of interest to declare.