ALBERT

Description: 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.

Description: Introduction: Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy; more than 90% are B cell lymphomas and express CD-20 antigen. Rituximab® (RTX) is an IgG1κ chimeric anti-CD20 mAb that binds specifically to the CD20-antigen on B lymphocytes. Our group previously reported that 99mTc-RTX represents a promising molecular imaging agent for NHL [1]. When 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/blood and tumor/normal tissue ratios with renal clearance. The development of radiolabeled Fab´s directed against specific targets may become a new strategy for NHL staging and surveillance. Objective: To radiolabel Fab´s (RTX) with 99mTc and to perform its chemical and biological evaluation. Methodology: We performed antibody fragmentation with papain and, once purified, fragments were identified by MaldiTOF/TOF and derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl2.2H2O was added to Fab´s (RTX)-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity was determined by HPLC. The in-vitro radiochemical stability of the radiolabeled Fab´s were analyzed in saline and serum up to 4 h. In-vitrobinding and competition assays were performed using Ramos and Raji cell lines up to 90 min. Biodistribution studies were evaluated in normal Balb/c mice and in Raji tumor-bearing Nude mice at 0.5 and 1 h. Results: Radiochemical purity of radiolabeled Fab´s were ≥90%. The in-vitro radiochemical stability studies showed that the radioconjugate was stable and no significant transchelation was detected. In-vitro binding and competition assays confirm that after its derivatization and radiolabeling, Fab´s (RTX) retained its specificity of binding to CD-20 antigen. This results confirm that Fab´s (RTX) affinity for CD20+ NHL cells remained unaffected after its derivatization. In-vivobiodistribution studies show that radiolabeled Fab´s has renal uptake with neglectable uptake in other organs, indicating that the primary route of clearance is renal. Lymph-node/muscle ratios of 4.00 and 2.55 at 0.5 and 1 h post injection, respectively. Conclusions: Fab´s (RTX) were easily and rapidly labeled demonstrating good stability and radiochemical purity. Based on lymph-node uptake and lymph-node/muscle ratios, 99mTc-HYNIC-Fab´s (RTX) may be useful for tumor molecular imaging agent for NHL. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: 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.