title: Radiolabeling of rituximab with (188)Re and (99m)Tc using the tricarbonyl technology.
abstract: BACKGROUND
  The most successful clinical studies of immunotherapy in patients with non-Hodgkin's lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20(+) B-cell tumors. Rituximab radiolabeled with β(-) emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the (99m)Tc- and (188)Re-tricarbonyl core (IsoLink technology).
  
  
  METHODS
  The native format of the antibody (RTX(wt)) as well as a reduced form (RTX(red)) was labeled with (99m)Tc/(188)Re(CO)(3). The partial reduction of the disulfide bonds to produce free sulfhydryl groups (-SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts.
  
  
  RESULTS
  The radiolabeling efficiency and kinetics of RTX(red) were superior to that of RTX(wt) ((99m)Tc: 98% after 3 h for RTX(red) vs. 70% after 24 h for RTX(wt)). (99m)Tc(CO)(3)-RTX(red) was used without purification for in vitro and in vivo studies whereas (188)Re(CO)(3)-RTX(red) was purified to eliminate free (188)Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37 °C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of (99m)Tc(CO)(3)-RTX(red) but not with pre-purified (188)Re(CO)(3)-RTX(red). Both conjugates revealed high binding affinity to the CD20 antigen (K(d) = 5-6 nM). Tumor uptake of (188)Re(CO)(3)-RTX(red) was 2.5 %ID/g and 0.8 %ID/g for (99m)Tc(CO)(3)-RTX(red) 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for (99m)Tc(CO)(3)-RTX(red) and for (188)Re(CO)(3)-RTX(red) 0.5 and 0.5 (24 h pi).
  
  
  CONCLUSIONS
  Rituximab could be directly and stably labeled with the matched pair (99m)Tc/(188)Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmunodiagnosis and therapy.
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, Av Professor Lineu Prestes 2242, 05508-000 São Paulo, Brazil.
authors.forename: Carla Roberta
authors.initials: CR
authors.lastname: Dias
authors.forename: Simone
authors.initials: S
authors.lastname: Jeger
authors.forename: João Alberto
authors.initials: JA
authors.lastname: Osso
authors.forename: Cristina
authors.initials: C
authors.lastname: Müller
authors.forename: Christine
authors.initials: C
authors.lastname: De Pasquale
authors.forename: Alexander
authors.initials: A
authors.lastname: Hohn
authors.forename: Robert
authors.initials: R
authors.lastname: Waibel
authors.forename: Roger
authors.initials: R
authors.lastname: Schibli
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000493:pharmacokinetics; D018951:Antigens, CD20 / Q000276:immunology; D001345:Autoradiography; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D004355:Drug Stability; D005260:Female; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008228:Lymphoma, Non-Hodgkin / Q000175:diagnosis / Q000378:metabolism / Q000532:radiotherapy; D051379:Mice; D015609:Organotechnetium Compounds / Q000737:chemistry*; D011868:Radioisotopes / Q000737:chemistry*; D012211:Rhenium / Q000737:chemistry*; D000069283:Rituximab
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D015609:Organotechnetium Compounds; D011868:Radioisotopes; D000069283:Rituximab; D006639:Histidine; D012211:Rhenium; D003545:Cysteine