title: Preparation & in vitro evaluation of ⁹⁰Y-DOTA-rituximab.
abstract: OBJECTIVE
  Radioimmunotherapy is extensively being used for the treatment of non-Hodgkin's lymphoma (NHL). Use of rituximab, a chimeric anti-CD20 antibody directed against the CD20 antigen in combination with suitable beta emitters is expected to result in good treatment response by its cross-fire and bystander effects. The present work involves the conjugation of p-isothiocyanatobenzyl DOTA (p-SCN-Bn-DOTA) to rituximab, its radiolabelling with [90] Y and in vitro and in vivo evaluation to determine its potential as a radioimmunotherapeutic agent.
  
  
  METHODS
  Rituximab was conjugated with p-SCN-Bn-DOTA at 1:1 antibody: DOTA molar ratio. The number of DOTA molecules linked to one molecule of rituximab was determined by radioassay and spectroscopic assay. Radiolabelling of rituximab with 90 Y was carried out and its in vitro stability was evaluated. In vitro cell binding studies were carried out in Raji cells expressing CD20 antigen. Biodistribution studies were carried out in normal Swiss mice.
  
  
  RESULTS
  Using both radioassay and spectroscopic method, it was determined that about five molecules of DOTA were linked to rituximab. Radiolabelling of the rituximab conjugate with [90] Y and subsequent purification on PD-10 column gave a product with radiochemical purity (RCP) > 98 per cent which was retained at > 90 per cent up to 72 h when stored at 37°C. In vitro cell binding experiments of 90 Y-DOTA-rituximab with Raji cells exhibited specific binding of 20.7 ± 0.1 per cent with [90] Y-DOTA-rituximab which reduced to 15.5 ± 0.2 per cent when incubated with cold rituximab. The equilibrium constant K d for 90 Y-DOTA-Rituximab was determined to be 3.38 nM. Radiolabelled antibody showed clearance via hepatobiliary and renal routes and activity in tibia was found to be quite low indicating in vivo stability of [90] Y-DOTA-rituximab.
  
  
  CONCLUSIONS
  p-SCN-Bn-DOTA was conjugated with rituximab and radiolabelling with 90 Y was carried out. In vitro studies carried out in Raji cells showed the specificity of the radiolabelled conjugate suggesting the potential uitability of the formulation as a radiopharmaceutical for therapy of NHL.
authors.affiliation: Isotope Production & Applications Division, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: Mythili
authors.initials: M
authors.lastname: Kameswaran
authors.forename: Usha
authors.initials: U
authors.lastname: Pandey
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Dash
authors.forename: Grace
authors.initials: G
authors.lastname: Samuel
authors.forename: Meera
authors.initials: M
authors.lastname: Venkatesh
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006571:Heterocyclic Compounds / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D051379:Mice; D009942:Organometallic Compounds / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D016499:Radioimmunotherapy / Q000379:methods*; D019275:Radiopharmaceuticals / Q000008:administration & dosage* / Q000737:chemistry; D000069283:Rituximab / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays
chemical_list: D006571:Heterocyclic Compounds; D009942:Organometallic Compounds; D019275:Radiopharmaceuticals; C113625:yttrium(III)-1,4,7,10-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid; D000069283:Rituximab