Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer.
Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with (99m) Tc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA(pos) LNCaP and PSMA(neg) PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. (99m) Tc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. (99m) Tc-PSMA30 internalized significantly more in LNCaP cells compared to (99m) Tc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for (99m) Tc-PSMA30 compared with (99m) Tc-PSMA6 and a (99m) Tc-control nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting.
Department of Immunology, Pasteur Institute of Iran, Tehran, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran.
Mehdi
M
Evazalipour
Matthias
M
D'Huyvetter
Bahram Soltani
BS
Tehrani
Mohsen
M
Abolhassani
Kobra
K
Omidfar
Shahriyar
S
Abdoli
Roghaye
R
Arezumand
Hamid
H
Morovvati
Tony
T
Lahoutte
Serge
S
Muyldermans
Nick
N
Devoogdt
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D043425:Glutamate Carboxypeptidase II / Q000037:antagonists & inhibitors* / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000378:metabolism; D019275:Radiopharmaceuticals; D061905:Single-Domain Antibodies; D013667:Technetium; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon; D014407:Tumor Cells, Cultured; D055114:X-Ray Microtomography; D023041:Xenograft Model Antitumor Assays
D000954:Antigens, Surface; D019275:Radiopharmaceuticals; D061905:Single-Domain Antibodies; D013667:Technetium; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II
PSMA; antibody fragment; imaging; nanobody