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111In/68Ga-Labeled anti-epidermal growth factor receptor, native chemical ligation cyclized Affibody ZHER2:342min.


Chopra A.


Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2013 Apr 03 [updated 2013 May 23].


The human epidermal growth factor receptor-2 (HER2, ErbB2) modulates its activity through a tyrosine kinase (TK) signaling pathway and is involved in the development of a variety of cancers (1, 2). Overexpression or amplification of the HER2 gene occurs in many cancer types (e.g., ~20% of breast cancer (BC)) and indicates a poor prognosis for the patient. Invasive methods such as biopsies, in conjunction with immunohistochemistry and in situ fluorescence hybridization, are often used to assess the HER2 status of the primary and metastasized neoplastic tumors; however, because of sampling bias and tumor heterogeneity, results obtained with these procedures are not completely reliable (2). In the clinic, 18F-labeled fluorodeoxyglucose is commonly used with positron emission tomography (PET) to detect and determine the tumor burden of a patient, but this imaging agent cannot distinguish between benign and malignant lesions, does not differentiate tumors that overexpress HER2 from those that have a low or no expression of the receptor, and often identifies inflammation as a false-positive neoplasm (1). Therefore, much effort has been devoted to produce and evaluate new imaging probes, such as radiolabeled anti-HER2 antibodies (3) and Affibodies (4), that can be used with single-photon emission computed tomography and PET to detect and diagnose cancers that overexpress HER2. An Affibody molecule is a chain of 58 amino acids (~6.5 kDa) that contains a modified B domain of the staphylococcal protein A and can be obtained either via chemical synthesis or produced in bacteria with recombinant DNA technology (5). The Affibody scaffold consists of 3-helix peptide chains: helix chains 1 and 2 are each composed of thirteen randomized amino acids and contain the receptor-binding moieties of the Affibody (6), and the third helix chain functions as a stabilizer of the Affibody molecule (7). Affibody molecules have a high affinity and specificity of binding to the target, such as a receptor, and are considered to be extremely suitable for the noninvasive imaging of solid tumors (8). The radionuclide-labeled Affibody ZHER2:342 (and its derivatives), directed against the HER2 (ZHER2:342Kd = 22 pM for HER2), has been used successfully with molecular imaging techniques to screen for BC patients who can probably benefit most from treatment with trastuzumab (a monoclonal antibody that targets HER2) or lapatinib (a small molecule drug that inhibits the TK activity of the HER2 signaling pathway) (8, 9). To further improve the imaging properties of radiolabeled Affibodies, it has been hypothesized that reducing the size of the molecule to a 2-helix structure will probably facilitate rapid clearance of the tracer from circulation, allow high penetration into solid tumors, ameliorate the uptake of label in non-targeted organs (such as the liver and lungs), and generate increased tumor/background ratios compared with the 3-helix molecule (10). To test this hypothesis, the third (stabilizing) helix chain was removed from the ZHER2:342 molecule; it was determined that the new 2-helix Affibody (MUT-DS; ~4.6 kDa) had a binding affinity of 5 nM for HER2 (10). In another study, 68Ga-labeled MUT-DS ([68Ga]-DOTA-MUT-DS) was evaluated for the detection of human ovarian carcinoma SKOV3 cell xenograft tumors, which overexpress HER2, in nude mice (11). Although the radioactivity from [68Ga]-DOTA-MUT-DS was rapidly cleared from circulation and had a high binding specificity for the tumors, the uptake of label in the neoplastic lesions was considerably lower (4.12 ± 0.83% of injected dose per gram tissue (ID/g) at 2 h postinjection (p.i.)) (11) than the accumulation with [68Ga]-ABY-002, another 68Ga-labeled HER2-binding 3-helix Affibody (12.4 ± 3.8% at 2 h p.i.) (12). This indicated that there was no particular advantage in using a smaller Affibody compared with the larger parent 3-helix molecule to detect tumors that overexpress HER2. The comparison between the two Affibody types was considered to be inaccurate because the data obtained with the two labeled Affibodies were generated in two separate and independent studies (6). In addition, the two SKOV3 cell subclones used to grow the tumors in mice in the two studies may not have produced identical lesions in the animals, and the same amounts of HER2 may not have been expressed in the neoplasms (6). Therefore, to ascertain the similarities or dissimilarities between MUT-DS and ABY-002 Affibodies, it was necessary to investigate the biological characteristics of the 2-helix and 3-helix Affibodies simultaneously using identical handling techniques and by performing similar in vitro and in vivo studies with the two Affibodies (6). The receptor targeting and biodistribution characteristics of 111In-labeled MUT-DS (denoted as [111In]PEP09239) and 111In-labeled ABY-002 ([111In]ABY-002) were investigated by Rosik et al. in a side-by-side study with mice bearing xenograft tumors (6). It was observed that although the accumulation of label from [111In]ABY-002 in the tumors was higher than with [111In]PEP09239, the tumor/blood ratio was higher with the latter tracer. This indicated that both Affibody types are suitable for the imaging of tumors that expressed HER2. However, these probes can be easily degraded by circulating exoproteases because they are unprotected at the terminals, and, due to the presence of disulfide bonds in their structures, only a limited number of methods can be used to radiolabel either Affibody (7). In a continuing effort to develop a smaller 2-helix scaffold, it was shown that a native chemical ligation technique can be used to produce such a molecule (denoted as Zmin) from the parental 2-helix Affibody (13). The characteristics of Zmin and the advantages of generating a molecular imaging probe based on the Zmin platform are discussed elsewhere (13). Hence, a backbone-cyclized version of the HER2-binding ZHER2:342 Affibody molecule, designated ZHER2:342min, was generated with the native chemical ligation technique and evaluated for its thermal stability, affinity, and selectivity for HER2 (7). Subsequently a DOTA-ZHER2:342min conjugate was prepared for labeling with 111In and 68Ga, and the tumor-targeting and biodistribution characteristics of [111In]ZHER2:342min and [68Ga]ZHER2:342min were compared with those of [111In]PEP09239 and [68Ga]PEP09239 (7).

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