title: Biodistribution of a (67)Ga-labeled anti-TNF VHH single-domain antibody containing a bacterial albumin-binding domain (Zag).
abstract: BACKGROUND
  Small domain antibodies (sdAbs) present high potential for both molecular in vivo imaging and therapy. Owing to the low molecular weight they are rapidly cleared from blood circulation, and new strategies to extend their half-lifes are needed for therapeutic applications. We have selected a bacterial albumin-binding domain (ABD) from protein Zag to be fused to an anti-tumor necrosis factor (TNF) single variable-domain heavy-chain region antibody (VHH) to delay blood clearance, and evaluated the biodistribution profile of the fusion protein.
  
  
  METHODS
  The anti-TNF VHH and the fusion protein VHH-Zag were conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). The anti-TNF and albumin-binding properties of the conjugates NOTA-VHH and NOTA-VHH-Zag were assessed by enzyme-linked immunosorbent assay (ELISA). The radioconjugates (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag were obtained by reaction of (67)GaCl3 with the corresponding conjugates at room temperature. Biodistribution studies were performed in healthy female CD-1 mice.
  
  
  RESULTS
  The immunoreactivity of the VHH-based proteins is preserved upon conjugation to NOTA as well as after radiometallation. The radiochemical purity of the radioconjugates was higher than 95% as determined by ITLC-SG after purification by gel filtration. The biodistribution studies showed that the Zag domain affected the pharmacokinetic properties of VHH, with impressive differences in blood clearance (0.028 ± 0.004 vs 1.7 ± 0.8 % I.A./g) and total excretion (97.8 ± 0.6 vs 25.5 ± 2.1 % I.A.) for (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag, respectively, at 24h p.i.
  
  
  CONCLUSIONS
  The Zag domain prolonged the circulation time of VHH by reducing the blood clearance of the labeled fusion protein (67)Ga-NOTA-VHH-Zag. In this way, the anti-TNF VHH in fusion with the Zag ABD presents a higher therapeutic potential than the unmodified VHH.
authors: Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139.7, 2695-066 Bobadela LRS, Portugal.
authors: Maurício
authors: M
authors: Morais
authors: CPM-URIA Fac. Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; IMM, Fac. Med., Universidade de Lisboa, Av. Prof. Egas Moniz 1649-028 Lisboa, Portugal.
authors: Cátia
authors: C
authors: Cantante
authors: Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139.7, 2695-066 Bobadela LRS, Portugal.
authors: Lurdes
authors: L
authors: Gano
authors: Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139.7, 2695-066 Bobadela LRS, Portugal.
authors: Isabel
authors: I
authors: Santos
authors: TechnoPhage S.A., Av. Prof. Egas Moniz, Edifício Egas Moniz Piso 2, 1649-028 Lisboa, Portugal.
authors: Sara
authors: S
authors: Lourenço
authors: CPM-URIA Fac. Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; IMM, Fac. Med., Universidade de Lisboa, Av. Prof. Egas Moniz 1649-028 Lisboa, Portugal.
authors: Catarina
authors: C
authors: Santos
authors: Fac. Med. Veterinária, Universidade de Lisboa, Av. da Universidade Técnica 1300-477 Lisboa, Portugal.
authors: Carlos
authors: C
authors: Fontes
authors: TechnoPhage S.A., Av. Prof. Egas Moniz, Edifício Egas Moniz Piso 2, 1649-028 Lisboa, Portugal.
authors: Frederico
authors: F
authors: Aires da Silva
authors: CPM-URIA Fac. Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; IMM, Fac. Med., Universidade de Lisboa, Av. Prof. Egas Moniz 1649-028 Lisboa, Portugal.
authors: João
authors: J
authors: Gonçalves
authors: Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139.7, 2695-066 Bobadela LRS, Portugal. Electronic address: jgalamba@itn.pt.
authors: João D G
authors: JD
authors: Correia
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D005260:Female; D005710:Gallium Radioisotopes / Q000627:therapeutic use; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D005710:Gallium Radioisotopes; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D014409:Tumor Necrosis Factor-alpha; C108290:ZAG protein, Streptococcal
keywords: Albumin-binding domain; Biodistribution; Gallium-67; Plasma half-life; Single-domain antibodies