In vitro analysis and in vivo tumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT.
Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes.


METHODS
We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and (99m)Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors.


RESULTS
The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with (99m)Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected (99m)Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys.


CONCLUSIONS
This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.
Vaneycken
Ilse
I
Laboratory for In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel (VUB), Brussels, Belgium. ilse.vaneycken@gmail.com
Govaert
Jochen
J
Vincke
Cécile
C
Caveliers
Vicky
V
Lahoutte
Tony
T
De Baetselier
Patrick
P
Raes
Geert
G
Bossuyt
Axel
A
Muyldermans
Serge
S
Devoogdt
Nick
N
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D002272:Carcinoembryonic Antigen / Q000276:immunology; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D017556:Technetium Compounds / Q000138:chemical synthesis; D014018:Tissue Distribution; D014055:Tomography, Emission-Computed; D015899:Tomography, Emission-Computed, Single-Photon
D002272:Carcinoembryonic Antigen; D002352:Carrier Proteins; D019275:Radiopharmaceuticals; D017556:Technetium Compounds