[PubMed]

The ¹⁸F-labeled N-(4-fluorobenzylidene)oxime (FBO)-monomeric Z_HER2:477 conjugate, abbreviated as ¹⁸F-FBO-Z_HER2:477, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1).

Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptide synthesis and to be cleared quickly by the kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody Z_HER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (Z_HER2:4)₂, exhibits an improved binding affinity of up to ~3 nM (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His₆-(Z_HER2:4)₂-Cys was generated by radiobrominating the dimeric (Z_HER2:4)₂ through the cysteine residues that were introduced to the C-terminus of (Z_HER2:4)₂ (7). Several affibody derivatives (e.g., ⁶⁸Ga-DOTA-Z_{HER2:342-pep2}, ¹¹¹In-DOTA-Z_{HER2:342-pep2}, ¹¹¹In-benzyl-DOTA-Z_HER2:342, and ¹¹¹In-benzyl-DTPA-Z_HER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the Z_HER2:342 peptide chain (3). Furthermore, these small affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody Z_HER2:342, which has an increased affinity of 50 nM (Z_HER2:4, the first generation) to 22 pM (8).

The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody Z_HER2:477 for molecular imaging. Both forms of the Z_HER2:477 molecule were radiofluorinated with an ¹⁸F-labeled prosthetic group of 4-¹⁸F-fluorobenzaldehyde (¹⁸F-FBO-Z_HER2:477 and ¹⁸F-FBO-(Z_HER2:477)₂, respectively) (1). The investigators have also coupled ⁶⁴Cu to the affibody through DOTA, leading to the development of imaging agents ⁶⁴Cu-DOTA- Z_HER2:477 and ⁶⁴Cu-DOTA-(Z_HER2:477)₂ (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors (refer to MUT-DS derived agents in MICAD).

This series of chapters summarizes the data obtained with the Z_HER2:477 derivatives, and this chapter presents the data obtained with ¹⁸F-FBO-Z_HER2:477 (1).

[PubMed]

The monomeric affibody Z_HER2:477 with a distal C-terminal cysteine residue (purity, >95%) is commercially available. The agent ¹⁸F-FBO-Z_HER2:477 was synthesized in three steps (1). The bifunctional linker 2-(aminooxy)-N-(2-(2,5-dioxo-2,5-dihydro-¹H-pyrrol-1-yl)ethyl)acetamide hydrochloride was first prepared by reacting N-(2-aminoethyl)malemide with 2-(tert-butoxycarbonylaminooxy)acetic acid under carbodiimide-mediated coupling conditions. The linker consisted of two orthogonal groups, a thiol-reactive maleimide group for conjugation to the cysteine residue and an ^18/19F-aldehyde–reactive aminooxy group. The yield was 80%. The linker was then selectively conjugated to the affibody molecule to generate aminooxy-functionalized Z_HER2:477 (Z_HER2:477-ONH₂; yield = 82%; molecular weight (MW) = 6,962.58 Da). As the last step, Z_HER2:477-ONH₂ was conjugated with 4-fluorobenzaldehyde (4-FBA), which resulted in the nonradioactive FBO-Z_HER2:477. The recovery yield was 70%–90%, and the measured MW was 7,068.94 Da for the nonradioactive FBO-Z_HER2:477. Similarly, conjugation of the Z_HER2:477-ONH₂ with 4-¹⁸F-FBA led to the generation of radioactive ¹⁸F-FBO-Z_HER2:477. For radiolabeling, the coupling yield was 30%–40%, and the overall radiochemical yield ranged from 13% to 18% (end of synthesis, corrected for decay). The specific activity and purity of ¹⁸F-FBO-Z_HER2:477 were 13.0–20.7 MBq/nmol (50–80 μCi/μg) and >95%, respectively, at end of synthesis. The total synthesis time was ~100 min.

[PubMed]

Binding affinity with the extracellular domain of HER2 antigen was measured in vitro with surface plasmon resonance detection (1). The measurements showed that the binding affinities of Z_HER2:477-ONH₂ and FBO-Z_HER2:477 were 400 and 430 pM, respectively (180 and 200 pM for the dimeric forms (Z_HER2:477)₂-ONH₂ and FBO-(Z_HER2:477)₂, respectively). The association constants were in the range of 10⁵–10⁶ (association time, >2 min), and the dissociation constants were in the range of 10^-4–10^-5 (disassociation time, >15 min), indicating that the on rate was very fast and the off rate was very slow.

The HER2-targeting ability for cultured cells was evaluated with SKOV3 human ovarian cancer cells (0.2 × 10⁶ cells/well) after incubation for 2 h with 1.11−6.29 kBq (0.03–0.17 μCi) ¹⁸F-FBO-Z_HER2:477. ¹⁸F-FBO-Z_HER2:477 quickly accumulated in the SKOV3 cells and reached the value of 0.00476 cell counts per minute [cpm]/medium cpm/μg of protein/μL at 0.5 h, which was maintained up to 2 h. In the presence of a large excess of nonradioactive Z_HER2:477 (final concentration, 0.28 μM), the cell uptake of ¹⁸F-FBO-Z_HER2:477 was inhibited significantly at all incubation time points (P < 0.05), showing a value of only 0.00329 from the unblocked amount of 0.00476 at 0.5 h, which suggests that the agent is specific to HER2 (1).

[PubMed]

No references are available.

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