Fig. 2.

(A–E) Site-specific ¹⁸F or ⁶⁴Cu labeling of single-domain antibodies (VHHs) using sortase. (A) A single-domain antibody fragment (VHH), equipped at its C terminus with the LPXTG sortase recognition motif followed by a His tag, is incubated with sortase A, which cleaves the threonine–glycine bond to yield the reactive thioacyl intermediate. Addition of a peptide with N-terminal glycine residues and a functional moiety of choice enables site-specific modification of the VHH. We thus modified a VHH with a (Gly)₃-tetrazine (Tz), as confirmed by LC-MS (liquid chromatography-mass spectrometry) (D, VHH7-Tz). (B) A tosyl-TCO and ¹⁸F-K222/K₂CO₃ were combined to produce ¹⁸F-TCO. (C) ¹⁸F-TCO was added to the Tz-modified VHH, and, after ∼20 min, the labeled VHH was retrieved by rapid size-exclusion chromatography. (E) The sortase reaction was used to install a NOTA functionality at the C terminus of a VHH followed by addition of ⁶⁴Cu²⁺ to produce ⁶⁴Cu-VHH. (F–M) ¹⁸F-VHH7 (anti-mouse class II MHC) detects secondary lymphoid organs. (F and G) PET-CT images of class II MHC^-/- (G) and C57BL/6 (F) mice 2 h postinjection of ¹⁸F-VHH7; numbers indicate (i) lymph nodes (numbers 1, 2, 3, 4, and 7), (ii) thymus (number 5), and (iii) spleen (number 6). (H and I) Coronal PET-CT images of C57BL/6 mouse imaged with ¹⁸F-VHH7, moving from anterior to posterior. In H and I, different sets of lymph nodes and thymus are visible. (K and L) Coronal PET-CT images of an MHC-II^-/- mouse imaged with ¹⁸F-VHH7, moving from anterior to posterior. In neither K nor L, lymph nodes or thymus is visible. (J and M) PET-CT as maximum intensity projections of all slices for a C57BL/6 and a class II MHC^-/- mouse 2 h postinjection of ¹⁸F-VHH7. In green, accumulation of ¹⁸F-VHH7 in lymph nodes and thymus. (PET scale bars have arbitrary units.) See Movies S1A, S1B, S2A, and S2B for 3D visualization of secondary lymphoid organs. (N and O) PET signals in vivo and postmortem biodistribution in all organs.