title: Preliminary evaluation of (177)Lu-labeled knottin peptides for integrin receptor-targeted radionuclide therapy.
abstract: OBJECTIVE
  Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts.
  
  
  METHODS
  Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for (177)Lu radiolabeling. The stability of the radiopeptides (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results.
  
  
  RESULTS
  DOTA-2.5D and DOTA-2.5F were labeled with (177)Lu at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to (177)Lu-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), (177)Lu-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of (177)Lu-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, (177)Lu-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of (177)Lu-DOTA-2.5D.
  
  
  CONCLUSIONS
  Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.
authors.affiliation: Department of Nuclear Medicine, Shanghai Ruijin Hospital, Shanghai Jiaotong University, 2nd Ruijin Rd, Shanghai, 200025, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Miao
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Hongguang
authors.initials: H
authors.lastname: Liu
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
authors.forename: Cathy S
authors.initials: CS
authors.lastname: Culter
authors.forename: Ande
authors.initials: A
authors.lastname: Bao
authors.forename: Peiyong
authors.initials: P
authors.lastname: Li
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D057771:Cystine-Knot Miniproteins / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000627:therapeutic use*; D015972:Gene Expression Regulation, Neoplastic; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008187:Lutetium / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011487:Protein Conformation; D011829:Radiation Dosage; D011854:Radiochemistry; D011868:Radioisotopes / Q000627:therapeutic use*; D011878:Radiotherapy / Q000379:methods*
chemical_list: D057771:Cystine-Knot Miniproteins; D006573:Heterocyclic Compounds, 1-Ring; D016023:Integrins; D010980:Platelet Membrane Glycoproteins; D011868:Radioisotopes; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D008187:Lutetium