[PubMed]

The ¹⁸F-labeled N-(4-fluorobenzylidene)oxime (FBO)-VENK[homoC]NKEMRNRYWEAALDPNLNNQQKRAKIRSIYDDP[homoC]-NH₂ (MUT-DS) conjugate with a disulfide bridge between the two homocysteines, abbreviated as ¹⁸F-FBO-MUT-DS, is a 2-helix affibody derivative that was synthesized by Miao et al. for positron emission tomography (PET) of HER2-expressing tumors (1).

Affibody molecules are a group of nonimmunogenic scaffold proteins that are derived from the B-domain of staphylococcal surface protein A (2, 3). These molecules have only 58 amino acid residues (~7 kDa), which form a 3-α-helical bundle structure (3, 4). Helices 1 and 2 bundles are responsible for the high binding affinity and specificity of affibodies to their targets, while helix 3 contributes to the affibody stabilization and is not involved in receptor recognition (4). Studies have further confirmed that the binding domain in the helices 1 and 2 bundles includes 13 amino acid residues that are surface-exposed (2). Therefore, large affibody libraries have been constructed by randomization of the 13 amino acid residues, and a large set of affibody molecules against a wide variety of targets have been selected from those libraries. Of them, the affibodies specific to HER2, including Z_HER2:342 and Z_HER2:477, have been intensively investigated in recent years (3, 5). These molecules have been radiolabeled and tested for molecular imaging of HER2-expressing tumors.

The investigators at Stanford University first tested the feasibility of the monomeric (~7 kDa) and dimeric (~14 kDa) forms of affibody Z_HER2:477 for molecular imaging (6, 7). Both forms have been labeled with various radiouclides through chelating agents. Studies have shown that smaller affibody constructs perform better in vivo in terms of tumor uptake and clearance, which prompts them to generate smaller proteins with only α-helices 1 and 2 bundles (~4 kDa) (4, 6, 7). However, simple deletion of the helix 3 leads to significantly decreased binding affinity of the proteins because of decreased helix conformation (4). The investigators then applied various strategies to improve the helix conformation of the affibody molecules, including sequence mutation, placement of disulfide bridges, and inclusion of helix-promoting amino acids (4). Although the helix conformation (~15%, the amount of α-helix represented in the secondary structure of the affibodies) of the 2-helix molecules is still much lower than that of the parent 3-helix affibodies, the investigators successfully obtained a class of 2-helix small proteins with HER2-binding affinity up to 5 nM with these strategies (1, 8, 9). 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 (4). The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for imaging HER2-expressing tumors. ⁶⁸Ga-DOTA-MUT-DS, ⁶⁴Cu-DOTA-MUT-DS, ¹¹¹In-DOTA-MUT-DS, and ¹⁸F-FBO-MUT-DS are examples of the MUT-DS derivatives (1, 8, 9).

This chapter summarizes the data obtained with ¹⁸F-FBO-MUT-DS (1).

[PubMed]

The linear aminooxy-functionalized (AO)-MUT-DS was synthesized with standard solid-phase peptide synthesis (purity, >95%) (1). Cyclization of the linear peptides was achieved with I₂ oxidation of the two l-homocysteines to form a disulfide bridge. The measured molecular weight (MW) of AO-MUT-DS was 4,823.9 (calculated 4,823.4). The AO-MUT-DS was then reacted with 4-fluorobenzaldehyde to prepare nonradioactive FBO-MUT-DS as a standard compound. Recovery yield was 70%–90%, and no starting cyclized peptide was detected. The measured MW (4,926.9) of the purified FBO-MUT-DS was consistent with the expected MW (4,928.4).

¹⁸F-FBO-MUT-DS was prepared by conjugating 4-¹⁸F-fluorobenzaldehyde with AO-MUT-DS (1). The purity was >95%. The overall yield ranged from 13% to 18% (non-decay-corrected) and the specific activities were 20–32 MBq/nmol (0.54–0.86 mCi/nmol) at the end of synthesis. The total time needed for the radiosynthesis was ~100 min.

[PubMed]

The binding affinities of AO-MUT-DS and FBO-MUT-DS with the extracellular domain of HER2 antigen were measured in vitro with surface plasmon resonance detection (1). The binding affinity of FBO-MUT-DS was slightly higher than that of AO-MUT-DS (2 nM and 1 nM, respectively). The off-rate of FBO-MUT-DS was found to be similar as the AO-MUT-DS with a dissociation constant of 7 × 10−⁴ 1/s. The on-rate of FBO-MUT-DS was only approximately one-fold higher than that of AO-MUT-DS, with association constants of 7.5 × 10⁶ 1 M/s (association time, >2 min).

The HER2-targeting ability for cultured cells was evaluated with SKOV3 human ovarian cancer cells over 1 h incubation with ¹⁸F-FBO-MUT-DS (1). ¹⁸F-FBO-MUT-DS quickly accumulated in SKOV3 cells and reached a value of 14% of applied activity at 0.25 h. The uptake was maintained at almost the same level until 1 h. When the probe was co-incubated with a large excess (final concentration 4 µg/ml) of nonradioactive Z_HER2:342, the probe uptake dropped to only ~5% of applied activity after 0.25 h incubation at 37°C (1).

[PubMed]

No references are available.

1.

Miao Z., Ren G., Jiang L., Liu H., Webster J.M., Zhang R., Namavari M., Gambhir S.S., Syud F., Cheng Z. A novel (18)F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor. Eur J Nucl Med Mol Imaging. 2011;38(11):1977–84. [PubMed: 21761266]

2.

Friedman M., Nordberg E., Hoiden-Guthenberg I., Brismar H., Adams G.P., Nilsson F.Y., Carlsson J., Stahl S. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. Protein Eng Des Sel. 2007;20(4):189–99. [PubMed: 17452435]

3.

Orlova A., Feldwisch J., Abrahmsen L., Tolmachev V. Update: affibody molecules for molecular imaging and therapy for cancer. Cancer Biother Radiopharm. 2007;22(5):573–84. [PubMed: 17979560]

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Webster J.M., Zhang R., Gambhir S.S., Cheng Z., Syud F.A. Engineered two-helix small proteins for molecular recognition. Chembiochem. 2009;10(8):1293–6. [PubMed: 19422008]

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Gao J., Chen K., Miao Z., Ren G., Chen X., Gambhir S.S., Cheng Z. Affibody-based nanoprobes for HER2-expressing cell and tumor imaging. Biomaterials. 2011;32(8):2141–8. [PMC free article: PMC3032351] [PubMed: 21147502]

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Cheng Z., De Jesus O.P., Kramer D.J., De A., Webster J.M., Gheysens O., Levi J., Namavari M., Wang S., Park J.M., Zhang R., Liu H., Lee B., Syud F.A., Gambhir S.S. 64Cu-labeled affibody molecules for imaging of HER2 expressing tumors. Mol Imaging Biol. 2010;12(3):316–24. [PubMed: 19779897]

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Cheng Z., De Jesus O.P., Namavari M., De A., Levi J., Webster J.M., Zhang R., Lee B., Syud F.A., Gambhir S.S. Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules. J Nucl Med. 2008;49(5):804–13. [PubMed: 18413392]

8.

Ren, G., J.M. Webster, Z. Liu, R. Zhang, Z. Miao, H. Liu, S.S. Gambhir, F.A. Syud, and Z. Cheng, In vivo targeting of HER2-positive tumor using 2-helix affibody molecules. Amino Acids, 2011.

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Ren G., Zhang R., Liu Z., Webster J.M., Miao Z., Gambhir S.S., Syud F.A., Cheng Z. A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression. J Nucl Med. 2009;50(9):1492–9. [PubMed: 19690041]