Radiosynthesis, optimization and pharmacokinetic study of the 99mTc-labeled human epidermal growth factor receptor 2 affibody molecule probe 99mTc-(HE)3ZHER2:V2 : Nuclear Medicine Communications

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Radiosynthesis, optimization and pharmacokinetic study of the 99mTc-labeled human epidermal growth factor receptor 2 affibody molecule probe 99mTc-(HE)3ZHER2:V2

Hu, Xianwen*,; Liang, Zhigang*,; Qi, Lili; Li, Fangming; Cai, Xiaojia; Cai, Jiong

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Nuclear Medicine Communications 44(4):p 244-251, April 2023. | DOI: 10.1097/MNM.0000000000001660



Human epidermal growth factor receptor 2 (HER2) is a protein encoded by the epidermal growth factor receptor family (ERBB)-2 gene in the human body, so it is also called receptor tyrosine-protein kinase ERBB-2 [1]. The structure of HER2 protein includes a tyrosine kinase domain in the cytoplasm, a transmembrane domain, and a ligand binding domain on the cell surface, of which carcinogenic mechanism includes inhibiting tumor cell apoptosis, promoting tumor cell proliferation, increasing tumor cell invasion, promoting tumor angiogenesis and lymphangiogenesis, and so on [2,3]. Studies have found that HER2 gene amplification and/or high expression of HER2 protein exist in a variety of malignant tumor tissues, including 60% of epithelial ovarian cancer, 35–45% of pancreatic cancer, 30% of breast cancer and 30% of 80% esophageal adenocarcinoma and squamous cell carcinoma [4–7]. Previous studies [8–10] have shown that the expression of HER2 protein is related to poor prognosis of tumors and the sensitivity of tumors to chemotherapy and biological therapy, so it is significant to accurately and effectively obtain HER2-positive expression for the precise diagnosis and targeted therapy of HER2-positive ovarian cancer.

Clinically, the status of HER2 is often determined by immunohistochemistry, nucleic acid fluorescence in situ hybridization and molecular imaging. In recent years, single-photon emission computed tomography (SPECT) and PET imaging can obtain functional information about tissue biochemistry, and become one of the important detection methods for studying HER2-positive lesions with the advantages of noninvasive, accurate and well-tolerated. It can not only obtain the HER2 positive expression of the whole body, including the location of the primary tumor, the degree of tumor invasion of surrounding tissues and the detection of metastases but also help doctors monitor the effect of targeted treatment of HER2 in patients, and contribute to the stratified study of tumor patients. Currently, commonly used molecular imaging probes targeting HER2 include antibodies, small molecular polypeptides, or proteins that specifically bind to the receptor [2]. Affibody is a kind of scaffold protein with moderate molecular weight, which has the advantages of high affinity with the target molecule, fast clearance in vivo and easy labeling [11,12]. As a radionuclide tracer for SPECT, 99mTc has a convenient source, low price and good nuclear physics properties, and its labeled molecular probe is suitable for SPECT imaging that is more widely carried out around the world. A variety of molecular probes of 99mTc-labeled HER2 affinity have been successfully developed and showed high affinity, but the high nonspecific uptake in the liver and kidney limits its clinical application [13–16]. The present study intends to remodel the amino terminus of the affibody ZHER2:V2 to synthesize a new HER2 affibody, to find the best 99mTc labeling conditions and to explore its potential application in HER2 receptor-positive tumors value.

Materials and methods

Design and synthesis of human epidermal growth factor receptor 2 affibody gene

By adding HEHEHE to the amino terminus of HER2 affibody ZHER2:V2, a new HER2 affibody (HE)3ZHER2:V2 was designed. Using the affibody gene sequence as a template, pET-26b(+) (purchased from Merck, USA) was used as an expression vector, and it was handed over to Sangon Bio-Company (ShangHai, China) for synthesis. The synthesized plasmid containing the affibody gene was double digested with NcoI and EcoRI (produced from BBI, UK) to verify the length of the synthesized gene; and gene sequencing was performed to verify the sequence of the synthesized gene.

Gene recombinant expression

The affibody pET-26b (+) plasmid was transformed into BL21 (DE3) (purchased from Merck, USA) Escherichia coli competent, and screened by Kana antibiotics (purchased from Sangsong, China) to obtain a monoclonal strain, then induced with Isopropyl β-D- Thiogalactopyranoside (IPTG) (purchased from Soleibo, China) and analyzed by SDS-PAGE gel electrophoresis to identify whether the affibody was expressed.

Affibody purification

E.coli expressing HER2 affibody was centrifuged at 8050 × g for 10 min. After centrifugation, the bacterial body weight was suspended in a buffer solution (50 mM Tris, 200 mM NaCl, PH 8.0), followed by ultrasonic crushing and centrifugation. The crushed supernatant was placed in a 60 °C water bath, then centrifuged, and the supernatant was taken and passed through a 0.45 mm filter membrane. The filtered liquid was taken, purified by an equilibrated Ni column, and eluted with a buffer containing 5, 15 and 60 mM imidazole for gradient elution and purification.

Affibody pretreatment

The 60 mmol/L imidazoline-eluted affibody was first dialyzed with PBS overnight, and 0.4 ml of the dialyzed affibody was added to DL-dithiothreitol (DTT) with a final concentration of 30 mmol/L and reduced in a constant temperature water bath at 37 °C for 1 h. Finally, the DTT was removed from reductive affibody by dialysis twice with ultrasonically degassed PBS, adding VitC to the affibody at a ratio of 0.1–0.3%, and labeled for use.

99mTc labeling and optimization of human epidermal growth factor receptor 2 affibody

Taking 200 μl of argon-deoxygenated labeling buffer (10 mmol/L N-2-hydroxyethylpiperazine -N' -2-ethanesulfonic acid, 20 mmol/L sodium glucoheptonate with pH 6.6), adding 100 µl affibody with a concentration of 1 g/L and 0.5 μl mixtures with a concentration of 50 g/L consisting of SnCl2·2H2O and HCl, distilled water. After blending, 100 µL 99mTcO4 (about 2 mCi) was added and placed in a metal bath at 25, 37, 50, 90 and 100 °C for 4–12 min for full reaction. Use a pipette to take a reaction mixture of 0.25 μl, and spot it at 1/5 of the thin-layer chromatography paper (ITLC-SG), and use PBS and citric acid as mobile phases to perform chromatography, and use a γ counter to measure the radioactivity count, calculate yield rate. According to the above experimental steps, different reaction conditions were set up respectively to find the best experimental conditions for 99mTc labeling HER2 affibody.

Purification of the 99mTc labeled human epidermal growth factor receptor 2 affibody

The radiochemical purity of 99mTc-labeled HER2 affibody was obtained through SDS-PAGE gel electrophoresis. After filtration by NAP-5 column, 20 µl of 99mTc-labeled HER2 affibody sample was added to 5 µl of protein loading buffer and then added to the gel well. After running the electrophoresis apparatus at 150 V for 30 min, the gel was removed from the splint and the radiochemical purity was calculated by measuring its radioactivity count with a γ counter.

Stability of the 99mTc labeled human epidermal growth factor receptor 2 affibody

The 99mTc labeled HER2 affibody was mixed with normal saline and human serum at a ratio of 1:9, respectively, in a warm bath at 37 °C. The radiochemical purity of 99mTc-(HE)3ZHER2:V2 was determined by thin layer chromatography with 0.25 μL of the above mixture at 0, 15, 30, 60, 120, 240 and 480 min, respectively.

The toxicity study of 99mTc-(HE) 3 Z HER2:V2

Eight 6-week-old Balb/C mice, weighing 18–22 g, were selected and randomly divided into two groups, including five in the experimental group and three in the control group, and their body weights were recorded respectively. Five Balb/C rats in the experimental group were injected with 99mTc-(HE)3ZHER2:V2 18–25 μCi (0.3 ml) from the tail vein. Three rats in the control group were injected with the same volume of sterile normal saline from the tail vein. During the experiment, mice were given sufficient water and food and received sufficient light during the day. The survival and activity of the mice were observed every day for 1 week. At the same time (12:00 noon) on the first, third, fifth and seventh days, the weights of the mice in the experimental group and the control group were compared before and after the experiment. After weighing the body weight on the seventh day, the mice were anesthetized with chloral hydrate, then sacrificed and dissected. The color and shape of the organs of the mice in the experimental group were observed and compared with those in the control group. The dissected organs and tissues were sent to the pathology department for HE staining, and the microscopic structures of the organs of the mice in the experimental group and the mice in the control group were observed.

Biodistribution of 99mTc-(HE) 3 Z HER2:V2in vivo

Twenty-four C57 mice (6–8 weeks old, 20.5 ± 1.8 g, purchased from Ensville Co., Ltd., Chongqing, China) were randomly divided into two groups. Mice in the experimental group were injected with 99mTc-(HE)3ZHER2:V2 with a concentration of 30.0 MBq/Kg and a volume of about 40–60 µl via the tail vein. The mice were killed 1, 2 and 4 h (three mice at each time point) after injection, dissected and tissue samples (blood, heart, liver, spleen, lung, kidney, stomach, intestine, bone, muscle, brain, thyroid and tumor) were extracted and weighed. To verify that adding the HEHEHE tag to the amino terminal of ZHER2:V2 (with the protein sequence AENKFNKEMRNAYWE IALLPNLTNQQKR AFIRSLYDDPS QSANLLAEAK KLNDAQGGGC) can optimize the biological distribution of molecular probe in vivo, 99mTc-ZHER2:V2 with the same radioactivity was injected into mice through the tail vein of mice and then dissected as the control group [The synthesis and 99mTc labeling methods of ZHER2:V2 are the same as those of (HE)3ZHER2:v2].

Pharmacokinetic study of 99mTc-(HE)3ZHER2:V2

Twenty-one C57 mice (6–8 weeks old, 20.0 ± 2.3 g, purchased from Ensville Co., Ltd, Chongqing, China) were used as spares. After the now-labeled 99Tc-(HE)3ZHER2:V2 was injected from the tail vein of the mice, blood was collected at 0, 10, 30 min, 1, 2, 4 and 24 h (three at each time point) after injection, respectively. At the corresponding time point, 0.2 ml of blood was collected from the mouse after enucleating the eyeball, and placed in a test tube, and the radioactivity count in each tube was measured with a γ counter. The radioactivity of blood was expressed as the percent injected dose rate per gram of tissue (%ID/g). The blood time-radioactivity curve was drawn, the compartment model was determined, and the pharmacokinetic parameters were calculated with DAS software version 2.0.


Design and synthesis of human epidermal growth factor receptor 2 affibody gene

A new HER2 affibody (HE)3ZHER2:V2 was designed with the protein sequence MAHEHEHEAENKFNK

EMRNAYW EIALLPNLT NQQKRAFIR SLYDDPSQ SANLLAEA KKLNDAQGGGC, and the coding sequence of the gene is N-atggCCCAT GAACAC GAGCACGAG GCGGAA AACAA ATTCAACAAA GAAATGC GCAACGCGT ACTGGGAAATTGCCCT GCTGCCGAACCTGACC AACCAACAGAAACGCG CCTTCATCCGCTCCCTG TACGACGACCCATCCCA ATCTGCAAACCTGCTGG CGGAAGCGAAGAAAC TGAACGATGCACAGGG TGGTGGTTGCTAA, of which theoretical molecular weight is about 7657.51. A fragment of about 200 bp was obtained by double digestion with endonuclease (NcoI) and EcoRI, which was consistent with the theoretical fragment size of 203 bp, indicating that the vector was successfully constructed (Fig. 1a), and gene sequencing confirmed that the synthesized gene sequence was completely consistent with the designed theoretical sequence (Fig. 1b).

Fig. 1:
Gene validation of affibody (HE)3ZHER2:V2. (a) NcoI and EcoRI digestion verification (i), DNA molecular scale, from top to bottom: 5K, 3K, 2K, 1.5K, 1K, 0.75K, 0.5K, 0.25K and 0.1K; (ii) digested samples); (b) Sequencing validation [(i), theoretical sequence; (ii), sequencing peak diagram]. NcoI, endonuclease.

Gene recombinant expression

The HER2 affibody (HE)3ZHER2:V2 plasmid cloned into pET-26b(+) was transformed into competent BL21(DE3) E. coli, induced by IPTG, the relative molecular weight of recombinant E. coli was found to be about 8 × 103 by SDS-PAGE analysis, which was consistent with the theoretical molecular weight of 7657.51, and a strain capable of expressing (HE)3ZHER2:V2 was obtained. The recombinantly expressed protein was purified by Ni affinity chromatography column, gradient elution was carried out, and the affibody was obtained in 60 mM imidazole (as shown in Fig. 2).

Fig. 2:
Recombinant expression and purification of (HE)3ZHER2:V2. 1, protein marker; 2, before induction; 3, after induction; 4, medium; 5, bacteria; 6, supernatant after ultrasound; 7, precipitation after ultrasound; 8, supernatant after water bath; 9, precipitation after water bath; 10, protein marker; 11, before column loading; 12, after column loading; 13, 5 mM imidazole wash; 14, 15 mM imidazole wash; 15, 60 mM imidazole elute.

Pr-treatment and 99mTc labeling of affibody

The dimeric affibody eluted by imidazole is reduced to monomer by DTT. The reaction mechanism of 99mTc labeled HER2 affibody (HE)3ZHER2:V2 is shown in Fig. 3. The HER2 affibody was combined with 99mTc under the action of buffer and SnCl2·2H2O, and its labeling yield was calculated by ITLC-SG thin-layer chromatography.

Fig. 3:
Schematic diagram of the reaction mechanism of 99mTc labeled HER2 affibody (HE)3ZHER2:V2.

The results of 99mTc labeling HER2 affibody are shown in Fig. 4, showing that the yield was the best when the reaction time was 6–10 min, and the labeling yield was 94.6% at 90 °C than at 25, 37 and 50 °C. The HER2 affibody was 200 μg with a concentration of 27.93 μmol/L, and the isotope technetium was 1 mCi with a radioactivity of 198.9 GBq/L, and the yield rate reached 95%. The labeling yield of using SnCl2·2H2O was higher than that of anhydrous SnCl2, and SnCl2·2H2O with a purity of 99.0% and a purity of 99.99% had little effect on the yield rate. The labeling yield of the mixture of SnCl2·2H2O and HCl showed little change when the concentration was 0.05–50 g/L, and the highest labeling rate was 95.6% when the concentration was 0.5 g/L. When the mixture of 50 g/L SnCl2·2H2O and HCl was diluted to 0.005 g/L, the labeling rate decreased obviously. When no SnCl2 or SnCl2·2H2O is added to the mixture, the labeling yield is extremely low and can be ignored. The labeling yield adding VitC to the reaction mixture, and using PBS as the mobile phase was slightly higher than that without adding VitC and using citric acid as the mobile phase. Using the NAP-5 column for purification, the radiochemical purity of 99mTc-(HE)3ZHER2:V2 was above 99.0%.

Fig. 4:
(a) The effect of reaction time and temperature on labeling yield; (b), The effect of the activity of affibody and isotope technetium on labeling yield; (c), The effect of SnCl2 on labeling yield; (d), The effect of VitC and developer (PBS or citric acid) on labeling yield.

Stability of the 99mTc labeled human epidermal growth factor receptor 2 affibody

99mTc-(HE)3ZHER2:V2 is quite stable in normal saline and human serum. When incubated at 37 °C for 2.0 h, its radiochemical purity is 94.2 and 93.5%, respectively, and the radiochemical purity remains above 90% within 8 h (as shown in Fig. 5).

Fig. 5.:
The stability of 99mTc-(HE)3ZHER2:V2 in normal saline and serum.

The toxicity study of 99mTc-(HE)3ZHER2:V2

During the experiment, the mice in the experimental group and the control group were alive and normal, and there were no significant differences in the structure, shape, color, body weight and microscopic structure of the two groups of mice and the control group (as shown in Supplementary Figure S1, Supplemental digital content 1,

Biodistribution of 99mTc-(HE)3ZHER2:V2 in vivo

The radioactivity uptake of 99mTc-(HE)3ZHER2:V2 and 99mTc-ZHER2:V2 in normal mouse liver at 1, 2 and 4 h were 1.52 ± 0.38 vs. 2.90 ± 0.84, 1.39 ± 0.32 vs. 2.75 ± 1.06 and 1.33 ± 0.43 vs. 2.50 ± 0.93, respectively. Compared with 99mTc-ZHER2:V2, besides the uptake of 99mTc-(HE)3ZHER2:V2 by the liver was significantly reduced, the uptake of radioactivity by the spleen and blood was also significantly reduced. The biodistribution of 99mTc-(HE)3ZHER2:V2 vs. 99mTc-ZHER2:V2 in the normal mice is depicted in Table 1.

Table 1 - Biodistribution of 99mTc-(HE)3ZHER2:V2 vs. 99mTc-ZHER2:V2in vivo
Tissues (%ID/g) Time (1 h) Time (2 h) Time (4 h)
99mTc-(HE)3ZHER2:V2 99mTc-ZHER2:V2 99mTc-(HE)3ZHER2:V2 99mTc-ZHER2:V2 99mTc-(HE)3ZHER2:V2 99mTc-ZHER2:V2
Heart 0.37 ± 0.06 0.54 ± 0.23 0.31 ± 0.10 0.28 ± 0.09 0.17 ± 0.05 0.35 ± 0.16
Liver 1.52 ± 0.38 2.90 ± 0.84 1.39 ± 0.32 2.75 ± 1.06 1.33 ± 0.43 2.50 ± 0.93
Spleen 0.22 ± 0.04 1.54 ± 0.56 0.25 ± 0.12 1.05 ± 0.37 0.20 ± 0.04 0.80 ± 0.34
Lung 0.55 ± 0.13 0.78 ± 0.22 0.34 ± 0.11 0.35 ± 0.06 0.32 ± 0.06 0.30 ± 0.12
Kidney 17.94 ± 5.34 15.68 ± 4.95 16.75 ± 5.08 12.68 ± 3.60 14.69 ± 3.90 10.05 ± 3.46
Brain 0.10 ± 0.02 0.40 ± 0.13 0.10 ± 0.04 0.12 ± 0.04 0.08 ± 0.02 0.10 ± 0.04
Muscle 1.60 ± 0.25 1.56 ± 0.72 1.30 ± 0.35 0.87 ± 0.29 1.11 ± 0.31 0.56 ± 0.17
Bone 0.55 ± 0.18 1.23 ± 0.45 0.45 ± 0.12 0.87 ± 0.32 0.45 ± 0.13 0.53 ± 0.10
Stomach 1.10 ± 0.34 2.43 ± 0.82 1.09 ± 0.31 1.54 ± 0.33 0.50 ± 0.05 1.04 ± 0.39
Duodenum 0.37 ± 0.12 1.06 ± 0.09 0.26 ± 0.08 0.96 ± 0.27 0.22 ± 0.01 0.79 ± 0.22
Thyroid 0.48 ± 0.12 0.96 ± 0.32 0.44 ± 0.10 0.83 ± 0.17 0.40 ± 0.05 0.89 ± 0.38
Blood 0.45 ± 0.11 1.84 ± 0.89 0.61 ± 0.20 0.97 ± 0.36 0.54 ± 017 0.86 ± 0.35
%ID/g: percent injected dose rate per gram tissue.

Pharmacokinetics of 99mTc-(HE)3ZHER2:V2

The drug-time curve and pharmacokinetic parameters of 99mTc-(HE)3ZHER2:V2 are shown in Fig. 6. The peak time was reached immediately after 99mTc-(HE)3ZHER2:V2 was intravenously injected into the mouse tail (Tmax = 0). The distribution half-life (T1/2α) is 0.047 ± 0.01 h, and the elimination half-life (T1/2β) is 0.851 ± 0.288 h. The peak concentration (Cmax) was 23.46 ± 6.831% ID/g, the mean residence time was 1.869 ± 0.157 h and the clearance rate was 2.08 ± 0.02%ID/g/h.

Fig. 6.:
(a) 99mTc-(HE)3ZHER2:V2 drug-time curve; (b) pharmacokinetic parameters of 99mTc-(HE)3ZHER2:V2.


HER2 affibody is a kind of scaffold protein with a small relative molecular mass obtained by phage display library screening, which can specifically bind to the HER2 receptor. HER2 affibody can be obtained by polypeptide synthesis and gene recombinant expression, but the cost of polypeptide synthesis is high. Compared to peptide synthesis, the method of recombinant expression synthetic affibody is simple and low in cost, and the amino- and carboxyl-terminus of the affibody can be modified by recombinant expression to facilitate purification and radionuclide labeling. Compared with antibody and polypeptide targeting molecular imaging probes, affinity targeting molecular imaging probes have moderate molecular weight, easy labeling, easy synthesis, high specificity, high affinity, and can be rapidly removed from the blood and nontargeted tissues, easy structural modification and low side effects [2,11,17]. In the current study, a new HER2 affibody (HE)3ZHER2:V2 was obtained by gene recombination by adding the HEHEHE sequence to the amino terminus of the HER2 affibody ZHER2:V2.

As a commonly used radionuclide for labeling SPECT imaging agents, 99mT has a half-life of 6.02 h, which can be obtained through a 99Mo-99mTc generator and is inexpensive, so it is widely used in clinical and basic research. At present, due to its nonspecific uptake in the liver, gastrointestinal tract, and so on, studies that the molecular probe of 99mTc-labeled HER2 affibody in ovarian cancer is still in the preclinical research stage [12,18]. Previous study revealed that the hydrophobicity of the HEHEHE tag can reduce the retention of the tracer in the liver [19]. Therefore, the design of this study is to introduce the HEHEHE tag at the n-terminal of the HER2 affibody ZHER2: V2, and our experimental results of biodistribution in normal mice are consistent with this view. Moreover, because the carboxyl terminal of the affibody ZHER2:V2 contains the GGGC sequence, which combines with the amide nitrogen of the adjacent amino acid and the thiol group of cysteine together form an N3S chelate structure, so as to achieve stable labeling of 99mTc without causing off-target [20]. The current research results show that the HER2 affibody (HE)3ZHER2:V2 can be labeled with the radionuclide 99mTc at room temperature, and the labeling process is simple, the labeling rate is high, and the labeling process takes less time, which can maintain high stability for a long time in serum and normal saline.

The pharmacokinetic study of 99mTc-(HE)3ZHER2:V2 in mice showed that the radiolabel distributes rapidly to the organs and tissues of the whole body (T1/2α = 0.047 ± 0.001 h). T1/2β is 0.851 ± 0.288 h, and it reflects the clearance rate of the drug from the body, which not only ensures that the target tissue (HER2-positive tumor tissue) is likely to have sufficient uptake time but also reveals that after the drug is injected into the body for about 50 min its radioactivity in the body is reduced by half, the background is reduced, and the target/nontarget ratio is increased, which will facilitate the clear imaging of the target tissue. These parameters indicate that 99mTc-(HE)3ZHER2:V2 has a suitable biological half-life and high in-vivo clearance rate required by radioactive imaging drugs, which will be beneficial to the smooth progress of imaging.

In conclusion, our study has successfully designed and synthesized a molecular probe 99mTc-(HE)3ZHER2:V2 for evaluating the expression status of HER2 receptors. The labeling process takes less time, and the labeling yield is high, with small toxic and side effects and a suitable half-life, so it is a SPECT molecular probe that is beneficial to the clear imaging of the target tissue. A limitation of the current study is that no further in-vivo imaging studies were performed in a nude mouse model of ovarian cancer, which will be needed in future studies.


This study was funded by the National Natural Science Foundation of the People’s Republic of China, NSFC (grant numbers: 81571712).

The data involved in the article can be obtained through the corresponding author under reasonable conditions.

J.C.: funding acquisition; Z.L.: investigation; X.C., F.L. and L.Q.: methodology; X.H.: writing-original draft; J.C.: writing- review and editing.

Conflicts of interest

There are no conflicts of interest.


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99mTc; affibody; human epidermal growth factor receptor; ovarian cancer; SPECT

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