First-in-human validation of enzymolysis clearance strategy for decreasing renal radioactivity using modified [68Ga]Ga-HER2 Affibody.

PURPOSE: Enzymolysis clearance strategy, characterized by releasing the non-reabsorbable radioactive fragment under the specific cleavage of enzymes, is confirmed to be a safe and effective way to reduce the renal radioactivity accumulation in mice. However, the effectiveness of this strategy in humans remains unknown. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various types of tumors, and radiolabeled HER2 Affibody is believed to be an attractive tool for HER2-targeted theranostics. However, its wide application is limited by the high and persistent renal uptake. In this study, we intend to validate the effectiveness of enzymolysis clearance strategy in reducing renal accumulation by using a modified HER2 Affibody. MATERIALS AND METHODS: A new HER2 Affibody ligand, NOTA-MVK-ZHER2:2891, containing a cleavable Met-Val-Lys (MVK) linker was synthesized and labeled with 68Ga. The microPET imaging study was performed in SKOV-3 tumor mice to assess the uptakes of the control ligand and the MVK one in tumors and kidneys. Seven healthy volunteers were included for biodistribution and dosimetric studies with both the control and MVK ligands performed 1 week apart. Urine and blood samples from healthy volunteers were collected for in vivo metabolism study of the two ligands. Four HER2-positive and two HER2-negative patients were recruited for [68Ga]Ga-NOTA-MVK-ZHER2:2891 PET/CT imaging at 2 and 4 h post-injection (p.i.). RESULTS: [68Ga]Ga-NOTA-MVK-ZHER2:2891 was stable both in PBS and in mouse serum. MicroPET images showed that the tumor uptake of [68Ga]Ga-NOTA-MVK-ZHER2:2891 was comparable to that of [68Ga]Ga-NOTA-ZHER2:2891 at all the time points, while the kidney uptake was significantly reduced 40 min p.i. (P < 0.05). The biodistribution study in healthy volunteers showed that the kidney uptake of MVK ligand was significantly lower than that of the control ligand at 1 h p.i. (P < 0.05), with the SUVmean of 34.3 and 45.8, respectively, while the uptakes of the two ligands in the other organs showed negligible difference. The effective doses of the MVK ligand and the control one were 26.1 and 28.7 µSv/MBq, respectively. The enzymolysis fragment of [68Ga]Ga-NOTA-Met-OH was observed in the urine samples of healthy volunteers injected with the MVK ligand, indicating that the enzymolysis clearance strategy worked in humans. The PET/CT study of patients showed that the range of SUVmax of HER2-positive lesions was 9.4-21, while that of HER2-negative lesions was 2.7-6.2, which suggested that the MVK modification did not affect the ability of ZHER2:2891 structure to bind with HER2. CONCLUSION: We for the first time demonstrated that enzymolysis clearance strategy can effectively reduce renal radioactivity accumulation in humans. This strategy is expected to decrease renal radiation dose of peptide and small protein-based radiotracers, especially in the field of radionuclide therapy.

Comparison of renal clearance of [18F]AlF-RESCA-HER2-BCH and [18F]AlF-NOTA-HER2-BCH in mice and breast cancer patients.

PURPOSE: A novel HER2 affibody-based molecular probe, [18F]AlF-RESCA-HER2-BCH, was developed for reducing renal uptake, evaluated, and compared with [18F]AlF-NOTA-HER2-BCH. METHODS: In preclinical studies, micro-PET/CT was performed using HER2-positive gastric cancer patient-derived xenografts (PDX) model at 0.5-1 (dynamic), 2, 4, and 6 h post-injection. For blocking experiment, 0.5 mg cold affibody was co-injected with probes. Biodistribution were performed on HER2-positive PDX models at 2 h post-injection. For clinical study, PET/CT images were acquired at 2 h and 4 h after injection of 231.29 ± 17.77 MBq [18F]AlF-NOTA-HER2-BCH or [18F]AlF-RESCA-HER2-BCH in five breast cancer patients (4 HER2-positive and 1 HER2-low). Standardized uptake values (SUVs) were measured in tumors and source-organs for semi-quantitative analysis. The OLINDA/EXM software (version 1.2) was used to calculate the radiation doses. RESULTS: [18F]AlF-NOTA-HER2-BCH and [18F]AlF-RESCA-HER2-BCH were stably labeled with [18F]F, with high binding specificity and affinity to HER2. Micro-PET/CT of both tracers could clearly visualize HER2-positive PDX tumors with high uptake of 16.24 ± 1.74% ID/g and 14.39 ± 2.45% ID/g at 2 h post-injection. The renal accumulation of [18F]AlF-RESCA-HER2-BCH was significantly lower than that of [18F]AlF-NOTA-HER2-BCH (5.16 ± 0.22% ID/g vs. 158.73 ± 5.44% ID/g at 2 h, p < 0.0001). In the clinical study, both [18F]AlF-NOTA-HER2-BCH and [18F]AlF-RESCA-HER2-BCH demonstrated favorable tumor targeting and image contrast. [18F]AlF-RESCA-HER2-BCH showed a higher SUVmax in both primary tumor and metastases, and a significantly higher target-to-nontarget ratio in metastases than [18F]AlF-NOTA-HER2-BCH. Moreover, [18F]AlF-RESCA-HER2-BCH had lower renal accumulation (43.56 ± 7.88 vs. 79.81 ± 3.81 at 2 h, p < 0.0001; 33.23 ± 6.89 vs. 78.63 ± 4.00 at 4 h, p < 0.0001) as well as a significantly lower renal absorbed dose than [18F]AlF-NOTA-HER2-BCH (0.4450 ± 0.1117 mGy/MBq vs. 0.8030 ± 0.1604 mGy/MBq, p < 0.01). CONCLUSIONS: [18F]AlF-RESCA-HER2-BCH tended to provide better image contrast than [18F]AlF-NOTA-HER2-BCH with a higher target-to-nontarget ratio in detection of metastases. Notably, [18F]AlF-RESCA-HER2-BCH had lower renal accumulation than [18F]AlF-NOTA-HER2-BCH.

Advances in the Application of Radionuclide-Labeled HER2 Affibody for the Diagnosis and Treatment of Ovarian Cancer.

Human epidermal growth factor receptor 2 (HER2) is a highly expressed tumor marker in epithelial ovarian cancer, and its overexpression is considered to be a potential factor of poor prognosis. Therefore, monitoring the expression of HER2 receptor in tumor tissue provides favorable conditions for accurate localization, diagnosis, targeted therapy, and prognosis evaluation of cancer foci. Affibody has the advantages of high affinity, small molecular weight, and stable biochemical properties. The molecular probes of radionuclide-labeled HER2 affibody have recently shown broad application prospects in the diagnosis and treatment of ovarian cancer; the aim is to introduce radionuclides into the cancer foci, display systemic lesions, and kill tumor cells through the radioactivity of the radionuclides. This process seamlessly integrates the diagnosis and treatment of ovarian cancer. Current research and development of new molecular probes of radionuclide-labeled HER2 affibody should focus on overcoming the deficiencies of non-specific uptake in the kidney, bone marrow, liver, and gastrointestinal tract, and on reducing the background of the image to improve image quality. By modifying the amino acid sequence; changing the hydrophilicity, surface charge, and lipid solubility of the affibody molecule; and using different radionuclides, chelating agents, and labeling conditions to optimize the labeling method of molecular probes, the specific uptake of molecular probes at tumor sites will be improved, while reducing radioactive retention in non-target organs and obtaining the best target/non-target value. These measures will enable the clinical use of radionuclide-labeled HER2 affibody molecular probes as soon as possible, providing a new clinical path for tumor-specific diagnosis, targeted therapy, and efficacy evaluation. The purpose of this review is to describe the application of radionuclide-labeled HER2 affibody in the imaging and treatment of ovarian cancer, including its potential clinical value and dilemmas.

Application of a Novel 68Ga-HER2 Affibody PET/CT Imaging in Breast Cancer Patients.

Background: Breast cancer is a heterogeneous disease, and the human epidermal growth factor receptor 2 (HER2) expression may vary considerably between primary and metastatic lesions, or even within a single lesion. Repeated biopsies cannot always be performed. In this feasibility trial, we assessed whether a novel 68Ga-NOTA-MAL-MZHER2 (68Ga-HER2) affibody PET/CT could determine the HER2 status of each lesion if there was a clinical need for it. Methods: 68Ga-HER2 affibody PET/CT was performed in breast cancer patients if HER2 status remained unclear after standard examinations (including bone scan, 18F-FDG PET/CT, CT, and feasible biopsy). All available images for each patient were evaluated through an independent review of two committee-certified radiologists with nuclear medicine expertise. In case of discrepancy, adjudication by a third radiologist was performed as needed. All radiologists were blinded to the clinical information. Results: Twenty-four patients were enrolled. 68Ga-HER2 affibody PET/CT was requested by physicians due to the following reasons: 6 with multiple primary cancers, 13 with metastases not amenable to biopsy or repeated biopsy, 6 with inconsistent HER2 status between primary and metastatic lesions, and 4 with different HER2 status within different metastases. The final PET report revealed that the 68Ga-HER2 affibody tumor uptake was considered positive in 16 patients, negative in 7 patients, and equivocal in one patient. The heterogeneity of 68Ga-HER2 affibody uptake was observed, with a maximal 8.5-fold difference within one patient and a maximal 11-fold difference between patients. 68Ga-HER2 affibody PET/CT demonstrated a high diagnostic accuracy in differentiating HER2-enriched breast cancer, with a sensitivity of 91.7% and a specificity of 84.6%, regardless of prior lines of anti-HER2 therapies. Conclusion: 68Ga-HER2 affibody PET/CT imaging could provide valuable information on HER2 expression of each tumor in the body of patients, which may help in personalized clinical decision-making. Its value is now under systemic assessment.

Improving Targeted Delivery and Antitumor Efficacy with Engineered Tumor Necrosis Factor-Related Apoptosis Ligand-Affibody Fusion Protein.

Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising protein candidate for selective apoptosis of a variety of cancer cells. However, the short half-life and a lack of targeted delivery are major obstacles for its application in cancer therapy. Here, we propose a simple strategy to solve the targeting problem by genetically fusing an anti-HER2 affibody to the C-terminus of the TRAIL. The fusion protein TRAIL-affibody was produced as a soluble form with high yield in recombinant Escherichia coli. In vitro studies proved that the affibody domain promoted the cellular uptake of the fusion protein in the HER2 overexpressed SKOV-3 cells and improved its apoptosis-inducing ability. In addition, the fusion protein exhibited higher accumulation at the tumor site and greater antitumor effect than those of TRAIL in vivo, indicating that the affibody promoted the tumor homing of the TRAIL and then improved the therapeutic efficacy. Importantly, repeated injection of high-dose TRAIL-affibody showed no obvious toxicity in mice. These results demonstrated that the engineered TRAIL-affibody is promising to be a highly tumor-specific and targeted cancer therapeutic agent.

Impact of 68Ga-NOTA-MAL-MZHER2 PET imaging in advanced gastric cancer patients and therapeutic response monitoring.

PURPOSE: Clinical PET imaging of human epidermal growth factor receptor 2 (HER2) can noninvasively detect HER2 overexpression in lesions. A novel 68Ga-NOTA-MAL-MZHER2 (68Ga-HER2) affibody was developed for clinical PET/CT, and its safety, tissue dosimetry, ability to detect HER2-positive lesions, and utility for HER2-targeted therapy in patients with advanced gastric cancer (AGC) were evaluated. METHODS: Thirty-four patients with AGC (23 with HER2-positive and 11 with HER2-negative primary lesions) were included and underwent PET/CT after an injection of approximately 3.7 MBq/kg body weight 68Ga-HER2 affibody. Thirteen patients (8 HER2-positive and 5 HER2-negative patients) were scanned at 1, 2, and 3 h post-injection to determine the best imaging timepoint, and the remaining patients were scanned at the optimized timepoint. All patients underwent standard 18F-FDG PET/CT within 7 d to identify viable lesions. The SUVmax of lesions larger than 1.0 cm were analyzed. Five lesion maxima were analyzed for each organ. RESULTS: (1) The 68Ga-HER2 affibody was safe and effective, and optimal image contrast was observed 2 h post-injection; the average effective absorbed dose was 0.0215 mSv/MBq. (2) The HER2-positive group had significantly higher 68Ga-HER2 affibody uptake than the HER2-negative group (SUVmax 10.7 ± 12.5 vs 3.8 ± 1.7, p = 0.005). The specificity and sensitivity were 100 and 55.4%, respectively, with a SUVmax cutoff value of 6.6. The SUVmax of the lesions ranged from 1.6 to 73.0, suggesting heterogeneity in HER2 expression. (3) 68Ga-HER2 affibody uptake showed an organ-dependent difference in patients with HER2-positive expression. Bone metastases had the highest uptake (SUVmax 40.5 ± 24.9), followed by liver metastases (SUVmax 11.9 ± 3.9) and lymph node metastases (SUVmax 5.6 ± 3.7), while the uptake in other lesions, including in the primary lesion, was relatively lower (SUVmax 7.3 ± 3.7). (4) Patients receiving therapy had a non-significantly lower lesion SUVmax than patients not receiving therapy (SUVmax 8.8 ± 4.9 vs 11.8 ± 15.2) (p = 0.253). Additionally, the 68Ga-HER2 affibody detected positive lesions in 1/11 patients with HER2-negative primary gastric cancer, which was confirmed by second generation gene sequencing. (5) Moreover, ten patients underwent baseline PET/CT followed by targeted anti-HER2 therapy. Patients with lesions showing high avidity to the 68Ga-HER2 affibody showed longer progression-free survival (PFS) than those with lesions showing low avidity (4-9 m vs 2-3 m). CONCLUSION: 68Ga-HER2 affibody PET/CT is a feasible method to noninvasively detect the HER2 status in AGC patients and enable early detection with a low dose. Ongoing anti-HER2 therapy did not influence 68Ga-HER2 affibody imaging, which allowed repeated evaluations to monitor the HER2 status after anti-HER2 therapy. This method provides an in vivo understanding of AGC biology that will ultimately help oncologists improve individualized therapy plans.

Improved therapeutic activity of HER2 Affibody-targeted cisplatin liposomes in HER2-expressing breast tumor models.

OBJECTIVES: The purpose of this study was to investigate whether the conjugation of anti-HER2-Affibody to cisplatin PEGylated liposome can efficiently enhance the therapeutic effectiveness of the targeted liposome. METHODS: First, Affibody molecules were incubated with Mal-PEG2000-DSPE micelle to afford formation of a maleimide-mediated thioether coupling to the COOH-terminal cysteine of Affibody. Cisplatin-loaded liposomes composed of hydrogenated soy phosphatidylcholine/ cholesterol/mPEG2000-DSPE (56.5:38.5:5 molar ratio) (150 mM) were prepared and characterized by their physicochemical properties. Affibody-conjugated micelles were then transferred into preformed liposomes by means of post insertion. The cytotoxicity and cellular uptake of Affibody-targeted (affisome) and nontargeted liposomes were tested in HER2(+) SK-BR-3, and the in vivo therapeutic activity was evaluated in TUBO breast cancer models. RESULTS: Anti-HER2 affisome demonstrated a higher amount of platinum intracellularly, and affected HER2(+)-SK-BR-3 cell death was at lower concentrations compared with its liposome counterparts. Further, cisplatin-affisome showed greater therapeutic efficiency than nontargeted liposome in HER2(+)-TUBO models. Equally promising, the affisome-treated mice did extend the survival of animals by several days and even left one tumor-free survivor. CONCLUSIONS: Affibody-targeting endowed cisplatin liposomes with significantly enhanced, albeit modest, therapeutic activity in HER2-overexpressing tumor model; however, further values are yet to be determined to advance clinical translation of these targeted nanoparticulates.

Active targeting using HER-2-affibody-conjugated nanoparticles enabled sensitive and specific imaging of orthotopic HER-2 positive ovarian tumors.

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER-2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.