UNASSIGNED: This study aimed to assess the biodistribution and bioactivity of the affibody molecular probe 99mTc-(HE)3ZHER2:V2, prepared by genetic recombination, and to investigate its potential for targeted human epidermal growth factor receptor 2 (HER2) imaging in SKOV3 ovarian cancer and MDA-MB-361 breast cancer xenografts.
UNASSIGNED: Affibody molecules were generated through genetic recombination. The radiochemical purity of the 99mTc-labeled HER2 affibody was determined using reverse phase high performance liquid chromatography (RP-HPLC). Evaluation of HER2 affinity in SKOV3 ovarian cancer cells and MDA-MB-361 breast cancer cells (HER2-positive) was conducted by calculating equilibrium dissociation constants. Biodistribution of the 99mTc-labeled affibody molecular probe was assessed in Balb/c mice bearing SKOV3 tumors. Tumor targeting specificity was evaluated in Balb/c mice using SKOV3, MDA-MB-361, and AT-3 (HER2-negative) xenografts.
UNASSIGNED: Affibody (HE)3ZHER2:V2, generated through recombinant gene expression, was successfully labeled with 99mTc, achieving a radiochemical purity of (96.0 ± 1.7)% (n = 3) as determined by RP-HPLC. This molecular probe exhibited specific binding to HER2-positive SKOV3 cells, demonstrating intense radioactive uptake. Biodistribution analysis showed rapid accumulation of 99mTc-(HE)3ZHER2:V2 in HER2-positive tumors post-administration, primarily clearing through the urinary system. Single-photon emission computed tomography imaging conducted 1-3 h after intravenous injection of 99mTc-(HE)3ZHER2:V2 into HER2-positive SKOV3 and MDA-MB-361 nude mouse models confirmed targeted uptake of the molecular probe by the tumors.
UNASSIGNED: The molecular probe 99mTc-(HE)3ZHER2:V2 developed in this study effectively targets HER2 for imaging HER2-positive SKOV3 and MDA-MB-361 xenografts in vivo. It exhibits rapid blood clearance without evident toxic effects, suggesting its potential as a valuable marker for detecting HER2 expression in tumor cells.

Epothilone B (Epo B), a promising antitumor compound effective against various types of cancer cells in vitro. However, its poor selectivity for tumor cells and inadequate therapeutic windows significantly limit its potential clinical application. Affibody is a class of non-immunoglobulin affinity proteins with precise targeting capability to overexpressed molecular receptors on cancer cells, has been intensively investigated due to its exceptional affinity properties. In this study, we present a targeted nanoagent self-assembled from the precursor of an affibody conjugated with Epo B via a linker containing the thioketal (tk) group that is sensitive to reactive oxygen species (ROS). The core-shell structure of the ZHER2:342-Epo B Affibody-Drug Conjugate Nanoagent (Z-E ADCN), with the cytotoxin Epo B encapsulated within the ZHER2:342 affibody corona, leads to significantly reduced side effects on normal organs. Moreover, the abundant presence of ZHER2:342 on the surface effectively enhances the targeting capacity and tumor accumulation of the drug. Z-E ADCN can be internalized by cancer cells via HER2 receptor-mediated endocytosis followed by Epo B release in response to high levels of ROS, resulting in excellent anticancer efficacy in HER2-positive tumor models.

Renal fibrosis is a representative pathological feature of various chronic kidney diseases, and efficient treatment is needed. Interstitial myofibroblasts are a key driver of kidney fibrosis, which is dependent on the binding of TGF-β1 to type I TGF-β receptor (TβRI) and TGF-β1-related signaling pathways. Therefore, attenuating TGF-β1 activity by competing with TGF-β1 in myofibroblasts is an ideal strategy for treating kidney fibrosis. Recently, a novel TβRI-mimicking peptide RIPΔ demonstrated a high affinity for TGF-β1. Thus, it could be speculated that RIPΔ may be used for anti-fibrosis therapy. Platelet-derived growth factor β receptor (PDGFβR) is highly expressed in fibrotic kidney. In this study, we found that target peptide Z-RIPΔ, which is RIPΔ modified with PDGFβR-specific affibody ZPDGFβR, was specifically and highly taken up by TGF-β1-activated NIH3T3 fibroblasts. Moreover, Z-RIPΔ effectively inhibited the myofibroblast proliferation, migration and fibrosis response in vitro. In vivo and ex vivo experiments showed that Z-RIPΔ specifically targeted fibrotic kidney, improved the damaged renal function, and ameliorated kidney histopathology and renal fibrosis in UUO mice. Mechanistic studies showed that Z-RIPΔ hold the stronger inhibition of the TGF-β1/Smad and TGF-β1/p38 pathways than unmodified RIPΔ in vitro and in vivo. Furthermore, systemic administration of Z-RIPΔ to UUO mice led to minimal toxicity to major organs. Taken together, RIPΔ modified with ZPDGFβR increased its therapeutic efficacy and reduced its systemic toxicity, making it a potential candidate for targeted therapy for kidney fibrosis.

Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.

In clinical practice, tumor-targeting diagnosis and immunotherapy against programmed death ligand 1 (PD-L1) have a significant impact. In this research, a PD-L1-antagonistic affibody dimer (ZPD-L1) was successfully prepared through Escherichia coli expression system, and conjugated with the photosensitizer of ICG via N-hydroxysuccinimide (NHS) ester to develop a novel tumor-targeting agent (ICG-ZPD-L1) for both tumor imaging diagnosis and photothermal-immunotherapy simultaneously. In vitro, ZPD-L1 could specifically bind to PD-L1-positive LLC and MC38 tumor cells, and ICG-ZPD-L1-mediated photothermal therapy (PTT) also showed excellent phototoxicity to these tumor cells. In vivo, ICG-ZPD-L1 selectively enriched into the PD-L1-positive MC38 tumor tissues, and the high-contrast optical imaging of tumors was obtained. ICG-ZPD-L1-mediated PTT exhibited a potent anti-tumor effect in vivo due to its remarkable photothermal properties. Furthermore, ICG-ZPD-L1-mediated PTT significantly induced the immunogenic cell death (ICD) of primary tumors, promoted maturation of dendritic cells (DCs), up-regulated anti-tumor immune response, enhanced immunotherapy, and superiorly inhibited the growth of metastatic tumors. In addition, ICG-ZPD-L1 showed favorable biosafety throughout the brief duration of treatment. In summary, these results suggest that ICG-ZPD-L1 is a multifunctional tumor-targeting drug integrating tumor imaging diagnosis and photothermal-immunotherapy, and has great guiding significance for the diagnosis and treatment of clinical PD-L1-positive tumor patients.

BACKGROUND: The chicken chorioallantoic membrane (CAM) model is a potential alternative to the mouse model based on the 3R principles. However, its value for determination of the in vivo behaviors of radiolabeled peptides through positron emission tomography (PET) imaging needed investigation. Herein, the chicken CAM tumor models were established, and their feasibility was evaluated for evaluating the imaging properties of radiolabeled peptides using a 68 Ga-labeled HER2 affibody.
METHODS: Two human breast cancer cell lines were inoculated into chicken CAM and mice, respectively. The tumor-targeting potential and pharmacokinetic profile of a 68 Ga-labeled affibody, 68 Ga-MZHER, in both tumor models were also determined.
RESULTS: The tumor-formation time in chicken CAM model was shorter than that of mouse model. The uptake values of human epithelial growth factor receptor-2 (HER2)-positive Bcap37 tumors in chicken CAM and mouse models were 5.36 ± 0.26% ID/g and 5.26 ± 0.43% ID/g at 30 min postinjection of 68 Ga-MZHER, respectively. At the same time points, the uptake values of HER2-negative MDA-MB-231 tumors in the chicken CAM models and mouse models were 1.57 ± 0.15% ID/g and 1.67 ± 0.25% ID/g, respectively. Ex vivo biodistribution confirmed that more radioactivity accumulated in Bcap37 tumors than in MDA-MD-231 tumors in both CAM and mouse models.
CONCLUSIONS: In this study, the CAM tumor model was successfully prepared. The chicken CAM model is a novel tool for quickly determining the in vivo properties of radiolabeled peptides targeting biomarkers. It may be beneficial for early monitoring of the therapeutic effect of a new drug through PET imaging with specific peptides.

The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of ZRBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.

BACKGROUND: The cancer-testis protein melanoma antigen A3 (MAGE-A3) is highly expressed in a broad range of malignant tumor forms. It has been confirmed that affibody molecules, a novel family of small (∼6.5 kDa) targeting proteins, are useful agents for molecular imaging and targeted tumor treatment. As a novel agent for in vivo molecular imaging detection of MAGE-A3-positive tumors, the efficacy of affibody molecules was assessed in this research.
METHODS: In this study, three cycles of phage display library screening resulted in the isolation of two new affibody molecules (ZMAGE-A3:172 and ZMAGE-A3:770) that attach to MAGE-A3. These molecules were then expressed in bacteria and purified. The affibody molecules with high affinity and specificity were evaluated using western blotting, immunohistochemistry, indirect immunofluorescence, surface plasmon resonance, and near-infrared optical imaging of tumor-bearing nude mice.
RESULTS: The selected ZMAGE-A3 affibodies can precisely bind to the MAGE-A3 protein in living cells and display high-affinity binding to the MAGE-A3 protein at the molecular level. Furthermore, the accumulation of DyLight755-labeled ZMAGE-A3:172 or ZMAGE-A3:770 in MAGE-A3-positive tumors was achieved as early as 30 min and disappeared at 48 h post-injection.
CONCLUSIONS: Our findings support the potential of the two MAGE-A3 protein-binding affibody molecules for their use as molecular imaging agents.

Antibody-drug conjugates (ADCs) play important roles in tumor therapy. However, traditional ADCs are limited by the extremely large molecular weight of the antibody molecules, which results in low permeability into solid tumors. The use of small ADCs may be expected to alleviate this problem, but this switch brings the new limitation of a greatly shortened blood circulation half-life. Here, we propose a new cleavable ADC design with excellent tumor tissue permeability and a long circulation half-life by fusing the small ADC ZHER2-MMAE with the Fc domain of the antibody for circulation half-life extension, and inserting a digestion sequence between them to release the small ADC inside tumors for better tumor penetration. The experimental results showed that the designed molecule Fc-U-ZHER2-MMAE has a significantly increased blood circulation half-life (7.1 h, 59-fold longer) compared to the small ADC ZHER2-MMAE, and significantly improved drug accumulation ability at tumor sites compared to the conventional full-length antibody-coupled ADC Herceptin-MMAE. These combined effects led to Fc-U-ZHER2-MMAE having significantly enhanced tumor treatment ability, as shown in mouse models of NCI-N87 gastric cancer and SK-OV-3 ovarian cancer, where Fc-U-ZHER2-MMAE treatment achieved complete regression of tumors in all or a portion of animals with no obvious side effects and an MTD exceeding 90 mg/kg. These data demonstrate the therapeutic advantages of this cleavable ADC strategy, which could provide a new approach for ADC design.

Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel-targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (ZTRI) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRβ). The aim of this study is to evaluate the feasibility of 68Ga-radiolabeled ZTRI ([68Ga]Ga-DOTA-ZTRI) as PET tracer for diagnosis of HCC.
The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRβ as HCC biomarker. The trimeric affibody ZTRI was conjugated with DOTA-NHS-ester and radiolabeled with 68Ga to produce [68Ga]Ga-DOTA-ZTRI conjugate. Immunoreactivity and specific uptake of [68Ga]Ga-DOTA-ZTRI were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [68Ga]Ga-DOTA-ZTRI PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [68Ga]Ga-DOTA-ZTRI in vivo.
Excessive PDGFRβ was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [68Ga]Ga-DOTA-ZTRI was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [68Ga]Ga-DOTA-ZTRI in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [68Ga]Ga-DOTA-ZTRI successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning.
Our results demonstrated that [68Ga]Ga-DOTA-ZTRI conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.