title: Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging.
abstract: BACKGROUND
  In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications.
  
  
  METHODS
  We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv).
  
  
  RESULTS
  Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a (111)In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model.
  
  
  CONCLUSIONS
  We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kelly Davis
authors.initials: KD
authors.lastname: Orcutt
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Slusarczyk
authors.forename: Maryelise
authors.initials: M
authors.lastname: Cieslewicz
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ruiz-Yi
authors.forename: Kumar R
authors.initials: KR
authors.lastname: Bhushan
authors.forename: John V
authors.initials: JV
authors.lastname: Frangioni
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D006241:Haptens / Q000276:immunology; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D016499:Radioimmunotherapy / Q000379:methods*; D011868:Radioisotopes / Q000737:chemistry / Q000627:therapeutic use*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D002614:Chelating Agents; D006241:Haptens; D006573:Heterocyclic Compounds, 1-Ring; D011868:Radioisotopes; D057127:Single-Chain Antibodies; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid