Generation of Site-specific labeled Nanobodies and the Validation for Molecular Imaging (2020)

66 Objectives: Nanobodies, monomeric domain of ~15 kDa proteins derived from camelid heavy-chain antibodies, retain excellent affinity and selectivity to target. Meanwhile, nanobodies have advantages over antibodies like rapid pharmacokinetics, better target-to-background contrast, high stability and weaker immunogenicity, making them attractive for molecular imaging and immunotherapy. However, the small size also renders challenges in their modification, since slight modification may cause a conformation change, leading to the loss of binding affinity towards antigens. Conventional methodology for conjugation produces heterogeneous mixtures which have random conjugation ratios and sites, which may make molecular imaging complicated. Herein, we examine the site-specific modification of an EGFR nanobody to study the Structure-Affinity-Relationship (SAR) by inserting an unnatural amino-acid (UAA) at a different site of the nanobody. We report here the in vitro and in vivo validation of this nanotechnology for molecular imaging and its translational potential.
 Methods: EGFR nanobody 10E8 was used as the model to produce site-specific nanobody-based tracer. BL21(DE3) stain was used for 10E8 expression. pSURAR-YAV plasmid, which contains the expanded genetic code system for an orthogonal amber suppressor tRNA-aaRS pair, was co-transfected with mutated 10E8 genes containing a TAG codon at certain sites for site-specifically incorporating UAA (NAEK). Western blot assay and mass spectrometry were used to confirm the successful incorporation of NAEK at the certain site. Surface plasmon resonance (SPR) assay was used to evaluate the binding affinity and selectivity of nanobodies to EGFR and non-target protein Her2. 10E8-82 was chose for further validation based on its excellent binding ability to EGFR, good selectivity and high expression yield. Immunofluorescence assay and FACS were used to confirm the binding ability and selectivity of 10E8-82. DIBO-Alex 488 was conjugated with 10E8-82 by a click reaction, followed by incubation with EGFR-positive expressed cell line SK-OV-3 or EGFR-negative expressed cells MCF-7. The binding ratio of 10E8-82 to SK-OV-3 and MCF-7 was analyzed. Biodistribution was determined by checking time dependent harvested tissue samples after tail vein injection with DIBO-Cy7 labeled 10E8-82. Dynamic imaging was performed in SK-OV-3 tumor bearing mice using IVIS SPECTRUM system (n = 6, Figure S1D).
 Results: 10E8 and mutated 10E8 with NAEK incorporation in residue R19, G26, T69, Q82, N84 were successfully generated, characterized by Western blot and mass spectrometry. According to SPR results, 10E8-82 and 10E8-84 showed comparable EGFR affinity (Kd = 4.80 and 4.80 nM) to 10E8 (Kd = 2.27 nM), meanwhile neither of them showed determinable affinity to Her2 (Figure S1B). Incorporation of NAEK in residue R19, G26 and T69 led to 2-5 fold loss of the affinity to EGFR, with Kd of 7.80, 18.60 and 9.24 nM, respectively. In FACS assay, 10E8-82 showed substantial targeting to EGFR-positive SK-OV-3 cells than EGFR-negative MCF-7 cells. The fraction of 10E8-82 binding to SK-OV-3 was 100%, whereas its binding to MCF-7 was only 5.79%. These results were further confirmed by confocal microscopy with clear staining of SKOV3 cells but no targeting was observed in MCF-7 cells (Figure S1C). In animal experiments, SK-OV-3 bearing mice were injected with Cy7-labeled 10E8-82, and images were obtained from 1 to 24 h post injection. 10E8-82 demonstrated a rapid tumor uptake as early as 1 h and reached a peak at 3 h post injection.
 Conclusions: We studied the SAR for the site-specific modification of a nanobody and the in vitro and in vivo validation by binding affinity and fluorescent imaging. The quick up-take of the target and fast clearance of nanobody makes it a promising candidate for fluorine-18 PET imaging, which is routinely used in clinic.
Acknowledgements: The studies were supported by NSFC 2017ZX09309009 and NIH/NIA K25AG061282.