Human epidermal growth factor receptor (HER2, erbB2, neu) is a 185-kDa transmembrane glycoprotein and is a member of the superfamily of epidermal growth factor receptor (EGFR)-type receptor tyrosine kinases (RTKs) (1, 2). HER2 consists of an extracellular domain with four subdomains, a single transmembrane span, a cytoplasmic juxtamembrane linker region, a tyrosine kinase component, and a carboxyl-terminal tail (2). HER2 forms homo-oligomers of itself and heteroligomers with other HER receptors to trigger a complex network of multilayered signal transduction (1), which involves more than 30 ligands and their related adaptor proteins, cascaded enzymes, second messengers, and transcription factors (3). Activation of the network regulates cell growth, cell differentiation, and cell survival. Overexpression of HER2 has been found in a variety of malignant tumors, such as breast cancers (~30% of patients), ovarian cancers, and urinary bladder cancers (4). No ligand that directly binds to HER2 has been clearly identified to date (4). Radionuclide imaging of breast cancers largely relies on radiolabeling of monoclonal antibodies directed against HER2, such as trastuzumab and pertuzumab (5). These antibodies are normally large in size (molecular weight ~150 kDa) and have slow blood clearance and slow tumor penetration, which lead to low contrast in images (6).
Affibody molecules are scaffold proteins that bind to targets with high affinity and specificity (7). Although Affibody molecules are able to bind to the same targets as immunoglobulins, Affibody molecules have no relation to the molecular structures or amino acid sequences of the immunoglobulin family. For example, ZHER2:342 (molecular weight ~7 kDa) is an Affibody molecule that specifically targets HER2, which consists of three-helix bundle Z-domains, each formed by 58 cysteine-free amino acids (8). The construction of ZHER2:342 is started with a three-helix bundle derived from the immunoglobulin-binding domain (B-domain) of staphylococcal protein A. Then the amino acids on the binding surface are replaced and randomized to remove the original binding affinity and create a completely new binding affinity. The randomization produces a library containing ~109 members, from which ZHER2:342 is identified as a ligand with high affinity to HER2 (22 pM). The helix bundle in ZHER2:342 provides structural rigidity and conformational stability for efficient binding to the target (9). Affibody molecules can be fused with other proteins/molecules to further modify their affinity/avidity, modulate in vivo kinetics, or introduce a peptide/protein effector function (6).
177Lu-CHX-A''-DTPA-ABD-Affibody (ZHER2:342)2 (177Lu-ABD-(ZHER2:342)2, molecular weight ~19 kDa) is used with single-photon emission computed tomography (SPECT) imaging of HER2 (5). 177Lu-ABD-(ZHER2:342)2 consists of a ZHER2:342 dimer, an albumin-binding domain (ABD), and a complex of 177Lu-[(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid) (177Lu-CHX-A''-DTPA). The use of dimeric ZHER2:342 allows for quick extravasation and tumor penetration and stabilizes ZHER2:342 binding to HER2 in the presence of ABD. The fused ABD (molecular weight ~5 kDa) is a stable, three-helix bundle of 46 amino acids derived from the monovalent variant of albumin-binding motif in streptococcal protein G (10). ABD binds to albumin in blood reversibly with a dissociation constant of 4 nM (10) to generate a complex of ~87 kDa, leading to a prolonged plasma half-life and reduced uptake in kidneys (5). 177Lu is a radionuclide belonging to the group of rare earth radionuclides, and it is produced by neutron bombardment of purified target material in reactors (11). With a half-life of 6.71 days for β- emission at 498 keV and 78% branch fraction, 177Lu has been a very promising radionuclide in radiotherapy for effectively destroying small tumors and metastasis (optimal size 1.2–3.0 mm) while sparing normal tissue (12). 177Lu also emits low-energy gamma rays at 208 and 113 keV with 10% and 6% abundance, respectively, which allows for direct monitoring of the activity distribution with SPECT and subsequent dosimetry (12).