title: Diabody Pretargeting with Click Chemistry In Vivo.
abstract: Radioimmunotherapy and nuclear imaging (immuno-PET/SPECT) of cancer with radiometal-labeled antibody fragments or peptides is hampered by low tumor-to-kidney ratios because of high renal radiometal retention. Therefore, we developed and evaluated a pretargeting strategy using click chemistry in vivo to reduce kidney uptake and avoid unwanted radiation toxicity. We focused on the bioorthogonal reaction between a trans-cyclooctene (TCO)-functionalized TAG72 targeting diabody, AVP04-07, and a low-molecular-weight radiolabeled tetrazine probe that was previously shown to have low kidney retention and relatively fast renal clearance.
  
  
  METHODS
  AVP04-07 diabodies were functionalized with TCO tags, and in vitro immunoreactivity toward bovine submaxillary mucin and tetrazine reactivity were assessed. Next, pretargeting biodistribution studies were performed in LS174T tumor-bearing mice with AVP04-07-TCO(n) (where n indicates the number of TCO groups per diabody) and radiolabeled tetrazine to optimize the TCO modification grade (0, 1.8, or 4.7 TCO groups per diabody) and the (177)Lu-tetrazine dose (0.1, 1.0, or 10 Eq with respect to the diabody). Radiolabeled tetrazine was injected at 47 h after diabody injection, and mice were euthanized 3 h later. A pretargeting SPECT/CT study with (111)In-tetrazine was performed with the optimized conditions.
  
  
  RESULTS
  Immunoreactivity for native AVP04-07 was similar to that for TCO-functionalized AVP04-07, and the latter reacted efficiently with radiolabeled tetrazine in vitro. The combination of the pretargeting component AVP04-07 functionalized with 4.7 TCO groups and 1 Eq of (177)Lu-tetrazine with respect to the diabody showed the most promising biodistribution. Specifically, high (177)Lu-tetrazine tumor uptake (6.9 percentage injected dose/g) was observed with low renal retention, yielding a tumor-to-kidney ratio of 5.7. SPECT/CT imaging confirmed the predominant accumulation of radiolabeled tetrazine in the tumor and low nontumor retention.
  
  
  CONCLUSIONS
  Pretargeting provides an alternative radioimmunotherapy and nuclear imaging strategy by overcoming the high renal retention of low-molecular-weight radiometal tumor-homing agents through the separate administration of a tumor-homing agent and a radioactive probe with fast clearance.
authors.affiliation: Tagworks Pharmaceuticals, Eindhoven, The Netherlands.
authors.forename: Sander M J
authors.initials: SM
authors.lastname: van Duijnhoven
authors.affiliation: Tagworks Pharmaceuticals, Eindhoven, The Netherlands Oncology Solutions, Philips Research, Eindhoven, The Netherlands.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Rossin
authors.affiliation: Precision and Decentralized Diagnostics, Philips Research, Eindhoven, The Netherlands; and.
authors.forename: Sandra M
authors.initials: SM
authors.lastname: van den Bosch
authors.affiliation: Avipep Pty. Ltd., Melbourne, Victoria, Australia.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Wheatcroft
authors.affiliation: Avipep Pty. Ltd., Melbourne, Victoria, Australia.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hudson
authors.affiliation: Tagworks Pharmaceuticals, Eindhoven, The Netherlands marc.robillard@tagworkspharma.com.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Robillard
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D045744:Cell Line, Tumor; D057930:Click Chemistry / Q000379:methods*; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging* / Q000532:radiotherapy; D005260:Female; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D058990:Molecular Targeted Therapy / Q000379:methods; D016719:Radioimmunodetection / Q000379:methods*; D016499:Radioimmunotherapy / Q000379:methods*; D019275:Radiopharmaceuticals / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000627:therapeutic use*; D016896:Treatment Outcome
chemical_list: D000911:Antibodies, Monoclonal; D019275:Radiopharmaceuticals; D057127:Single-Chain Antibodies
keywords: Diels–Alder; click; diabody; pretargeting