Search Results (83)

Introduction: Platelet-derived growth factor receptor beta (PDGFRβ) is a key regulator of fibrogenesis. Non-invasive imaging of PDGFRβ expression may offer a novel approach to assess fibrotic remodeling, particularly in cardiac patients’ post-intervention, where fibrosis poses clinical risk. This study presents the GMP-compliant production of a novel PDGFRβ-targeted PET radiopharmaceutical, [⁶⁸Ga]Ga-DOTA-Z09591 ([⁶⁸Ga]Ga-ATH001), and its preclinical evaluation in mouse and human myocardial tissue, along with initial clinical imaging in patients with ST-elevation myocardial infarction (STEMI). Methods: The precursor was chemically synthesized and radiolabeled with gallium-68 using a fully automated, GMP-compatible system and a pharmaceutical-grade ⁶⁸Ge/⁶⁸Ga generator. Autoradiography, H&E, Sirius Red, Masson’s trichrome, and IHC staining were performed on infarcted mouse hearts and human myocardial biopsies. In vivo PET/MRI with [⁶⁸Ga]Ga-ATH001, ¹⁵O-H₂O, and gadolinium contrast was conducted in STEMI patients one week post-percutaneous coronary intervention. Results: [⁶⁸Ga]Ga-ATH001 was produced with high radiochemical yield and purity. Autoradiography demonstrated specific, receptor-mediated binding of [⁶⁸Ga]Ga-ATH001, co-localizing with PDGFRβ immunoreactivity, collagen deposition, and tissue damage. In STEMI patients, focal tracer uptake was observed in infarcted myocardium correlating with MRI-detected structural abnormalities and perfusion defects on ¹⁵O-H₂O PET. Uptake in unaffected myocardium was low and homogeneous, consistent with minimal physiological PDGFRβ expression. Conclusions: [⁶⁸Ga]Ga-ATH001 was successfully developed and validated for phase 0 clinical study. The tracer demonstrated PDGFRβ-specific binding in human fibrotic myocardium and enabled non-invasive detection of myocardial fibrogenic activity in STEMI patients. These findings support further clinical evaluation of [⁶⁸Ga]Ga-ATH001 as a targeted molecular imaging agent for early assessment of post-infarction fibrosis. Full article

Affibody molecules have emerged as versatile protein engineering platforms due to their exceptional binding properties. These small (6.5 kDa) three-helix bundle proteins, derived from the Z-domain of Staphylococcal protein A, can be engineered to bind diverse molecular targets with high affinity and specificity. This structural and functional versatility has driven their applications in diagnostics, therapeutics, and biosensing. This review examines the evolution from monomeric affibody constructs to multivalent supramolecular assemblies, highlighting how this shift overcomes key limitations while expanding functionality. Recent advances in conjugation chemistry, scaffold engineering, and protein design have enabled sophisticated affibody-based architectures with enhanced pharmacokinetic profiles and multivalent binding capabilities, thereby improving their utility in targeted drug delivery, molecular imaging, and theranostics. Full article

Photorhabdus luminescens produces a syringe-like toxin complex to inject toxic enzymes into cells. We demonstrated that the recombinant Photorhabdus toxin complex (PTC) can be engineered for translocation of foreign cargo proteins across cellular membranes. We showed that the system is suitable for injection of trimeric affibodies into mammalian cells in order to influence crucial signaling pathways. As proof of principle, we inhibited Ras-driven tumor cell proliferation by injection of an affibody which interacts with the Ras binding domain of Raf kinase. The system described here could be applicable to target a wide range of signaling molecules for cell biological or therapeutic intervention. Full article

The TMEM16 (Anoctamin) family comprises a group of transmembrane proteins involved in diverse physiological processes, including ion transport and phospholipid scrambling. TMEM16F (Anoctamin 6), a phospholipid scramblase and nonselective ion channel, plays a central role in membrane remodeling, blood coagulation, immune responses, and cell death pathways through its ability to externalize phosphatidylserine in response to elevated intracellular calcium levels. Consequently, modulating TMEM16F activity has emerged as a promising strategy for the development of new therapeutic applications. Despite the functional importance of TMEM16F, TMEM16F modulators have received little study. In a previous study, we generated TMEM16F-specific affibodies by biopanning a phage display library for affibodies that bind to brain-specific TMEM16F (hTMEM16F) variant 1. In this study, we selected six other affibodies from among the 38 previously sequenced affibody candidates and characterized them. After purification, we confirmed that two of these affibodies bound to human TMEM16F with high affinity. To provide functional insights into how these affibodies modulate TMEM16F activity, we tested whether they could exert functional effects at the cellular level. Finally, we show that TMEM16F affibody attenuated the neuronal cell death induced by glutamate and microglial phagocytosis, suggesting that these affibodies might have potential therapeutic and diagnostic applications. Full article

Positron emission tomography (PET) imaging targeting glypican-3 (GPC3) holds promise for improving the detection and characterization of hepatocellular carcinoma (HCC). Preclinical and early clinical studies have largely utilized high-molecular-weight antibodies radiolabeled with isotopes such as ⁸⁹Zr and ¹²⁴I, demonstrating high affinity and tumor uptake but suffering from prolonged circulation times and suboptimal signal-to-background ratios. To address these limitations, interest has shifted toward low-molecular-weight vectors—synthetic peptides and small antibody fragments—labeled with shorter-lived radionuclides (e.g., ⁶⁸Ga and ¹⁸F) to enable rapid pharmacokinetics and same-day imaging protocols. Emerging platforms such as affibodies and aptamers offer further advantages in target affinity and reduced immunogenicity. However, clinical translation requires rigorous validation: larger, histologically confirmed cohorts, head-to-head comparison with CT/MRI, and correlation with hard clinical endpoints. Moreover, leveraging GPC3 expression as a biomarker could guarantee a deeper knowledge of tumor biology—differentiation grade and vascular invasion risk—and guide theranostic strategies. While β-emitters (⁹⁰Y, ¹⁷⁷Lu) have been explored for GPC3-directed therapy, their efficacy is influenced by oxygenation and cell-cycle status, whereas α-emitters (²²⁵Ac) may overcome these constraints, albeit with challenges in radionuclide selection and daughter nuclide management. Finally, dual-targeting probes combining GPC3 and prostate-specific membrane antigen (PSMA) have demonstrated superior uptake and retention in murine models, suggesting a versatile approach for future clinical diagnostics and therapy planning. Full article

Background: The ⁸⁹Zr radioisotope is increasingly vital in positron emission tomography (PET), especially immuno-PET, due to its long half-life of 78.4 h, allowing extended tracking of biological processes. This makes it particularly suitable for researching medicines with slow pharmacokinetics and enhances the precision of molecular imaging, especially in oncology. Despite zirconium’s potential for skeletal accumulation, effective chelation with agents like deferoxamine (DFO) enables high-resolution imaging of antigen-specific tumours, such as HER2-positive breast cancer, offering insights into tumour biology and treatment response. Methods: ⁸⁹Zr was produced at the ACSI TR-19 cyclotron via ⁸⁹Y(p,n)⁸⁹Zr reaction. Natural yttrium foils (250 μm) were irradiated with 12.9 MeV protons on target, with 100 μA·h. An HER2-targeting affibody was synthesized and conjugated with p-NCS-Bz-DFO (1:4 mass ratio) at 37 °C for 60 min (pH 9.2 ± 0.2), then purified on a PD-10 column. Radiolabelling was performed with [⁸⁹Zr]Zr-oxalate at pH ranging from 7.0 to 9.0, with concentrations from 110 to 460 MBq/mL. Results: Final activity reached 2.95 ± 0.31 GBq/batch (EOB corrected), with ≥ 99.9% radionuclide and ≥95% radiochemical purities. The anti-HER2 affibody was successfully radiolabelled with ⁸⁹Zr, resulting in a radiochemical purity of over 85% with molar activity of 26.5 ± 4.4 and 11.45 MBq/nmol at pH 7.0–7.5. In vitro tests on BT-474 and MCF-7 cell lines confirmed high uptake in HER2-positive cells, validating specificity and stability. Conclusions: The successful synthesis and labelling of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody are promising achievements for its further application in targeted immuno-PET imaging for HER2-positive malignancies. Further in vivo studies are needed to support its clinical translation. Full article

Background/Objectives: Biodegradable polymers have emerged as promising platforms for drug delivery. Produced by microbiomes, polyhydroxyalkanoates (PHAs) offer excellent biocompatibility, biodegradability, and environmental sustainability. In this study, we report the surface functionalization of PHA-based nanoparticles (NPs) using the SpyTag–SpyCatcher system to enhance cellular uptake. Methods: Initial conjugation with mEGFP-SpyTag enabled visualization, followed by decoration with HER2-specific Affibody-SpyCatcher and/or TAT-SpyCatcher peptides. The prepared NPs retained a diameter of <200 nm and a negatively charged surface. Results: Affibody-functionalized NPs significantly enhanced internalization and cytotoxicity in HER2-overexpressing SK-BR-3 cells, whereas TAT-functionalized NPs promoted uptake across various cell types, independently of HER2 expression. Dual-functionalized NPs exhibited synergistic or attenuated effects based on the HER2 expression levels, highlighting the critical role of ligand composition in targeted delivery. Conclusions: The results of this study demonstrate that the SpyTag–SpyCatcher-mediated surface engineering of PHA NPs offers a modular and robust strategy for active targeting in nanomedicine. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer treatment that uses near-infrared light to activate a conjugate of a monoclonal antibody (mAb) and a photoactivatable silica phthalocyanine dye (IRDye700DX: IR700). Unlike conventional photodynamic therapy (PDT), NIR-PIT selectively destroys targeted tumor cells while preserving the surrounding normal tissue and providing superior tissue penetration. Recently, NIR-PIT has been approved for the treatment of unresectable recurrent head and neck cancers in Japan. It induces highly selective cancer cell death; therefore, it is expected to be a new curative treatment option for various cancers, including brain tumors. In this review, we compare the principles of NIR-PIT and PDT and discuss the potential applications of NIR-PIT for brain tumors. We selected targetable proteins across various types of brain tumors and devised a strategy to effectively pass the mAb–IR700 conjugate through the blood–brain barrier (BBB), which is a significant challenge for NIR-PIT in treating brain tumors. Innovative approaches for delivering the mAb–IR700 conjugate across the BBB include exosomes, nanoparticle-based systems, and cell-penetrating peptides. Small-molecule compounds, such as affibodies, are anticipated to rapidly accumulate in tumors within intracranial models, and our preliminary experiments demonstrated rapid uptake. NIR-PIT also induces immunogenic cell death and activates the anti-tumor immune response. Overall, NIR-PIT is a promising approach for treating brain tumors. It has the potential to overcome the limitations of conventional therapies and offers new hope to patients with brain tumors. Full article

Nasopharyngeal carcinoma (NPC) is a tumor of the head and neck, with a higher incidence in southern China and Southeast Asia. Radiotherapy and chemotherapy are the main treatments; however, metastasis and recurrence remain the main causes of treatment failure. Further, the majority of patients are diagnosed in the late stage due to lack of tumor-specific biomarker for early diagnosis. Therefore, an effective treatment and early detection can improve the outcome of patient with NPC. Axl and EGFR are co-expressed in NPC tissues and play key roles in tumor proliferation, migration, and invasion, which are often correlated with poor prognosis and therapy resistance. In this study, we generated a novel bispecific affibody (Z_239-1907) for the dual targeting and inhibition of Axl and EGFR expression in NPC-positive cells both in vitro and in vivo. The in vitro experiments demonstrated that Z_239-1907 had more pronounced antitumor effects than either modality alone (Z_AXL239 or Z_EGFR1907) in NPC-positive cells. Further, mice bearing NPC-positive tumors showed significant inhibition in tumor growth after treatment with Z_239-1907 compared to Z_AXL239 and Z_EGFR1907. The in vivo tumor targeting ability and imaging also showed that Z_239-1907 specifically and selectively targeted NPC xenograft mice models and accumulate at tumor site as early as 30 min and disappeared within 24 h post-injection. Collectively, these results suggest that Z_239-1907 dual-target affibody is a promising therapeutic agent and a molecular imaging probe for early diagnosis in NPC. Full article

The combination of paclitaxel (PTX) with other chemotherapy drugs (e.g., gemcitabine, GEM) or genetic drugs (e.g., siRNA) has been shown to enhance therapeutic efficacy against tumors, reduce individual drug dosages, and prevent drug resistance associated with single-drug treatments. However, the varying solubility of chemotherapy drugs and genetic drugs presents a challenge in co-delivering these agents. In this study, nanoparticles loaded with PTX were prepared using the biodegradable polymer material poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). These nanoparticles were surface-modified with target proteins (Affibody molecules) and RALA cationic peptides to create core-shell structured microspheres with targeted and cationic functionalization. A three-drug co-delivery system (PTX@PHBHHx-ARP/siRNA_GEM) were developed by electrostatically adsorbing siRNA chains containing GEM onto the microsphere surface. The encapsulation efficiency of PTX in the nanodrug was found to be 81.02%, with a drug loading of 5.09%. The chemogene adsorption capacity of siRNA_GEM was determined to be 97.3%. Morphological and size characterization of the nanodrug revealed that PTX@PHBHHx-ARP/siRNA_GEM is a rough-surfaced microsphere with a particle size of approximately 150 nm. This nanodrug exhibited targeting capabilities toward BT474 cells with HER2 overexpression while showing limited targeting ability toward MCF-7 cells with low HER2 expression. Results from the MTT assay demonstrated that PTX@PHBHHx-ARP/siRNA_GEM exhibits high cytotoxicity and excellent combination therapy efficacy compared to physically mixed PTX/GEM/siRNA. Additionally, Western blot analysis confirmed that siRNA-mediated reduction of Bcl-2 expression significantly enhanced cell apoptosis mediated by PTX or GEM in tumor cells, thereby increasing cell sensitivity to PTX and GEM. This study presents a novel targeted nanosystem for the co-delivery of chemotherapy drugs and genetic drugs. Full article

Background: Affibody molecules represent a class of highly specific binders of particular interest for the development of highly affine target-specific radiopharmaceuticals. Their chemical synthesis is, however, intricate due to their considerable length of 58 amino acids; thus, approaches to optimize their preparation are constantly being sought. Methods: As ultrasound assistance has recently been shown to increase the efficiency of amino acid conjugation during solid-phase peptide synthesis (SPPS), the influence of ultrasonication on the outcome of the SPPS-based preparation of the EGFR-specific affibody Z_EGFR:1907 was compared to a common protocol relying on mechanical shaking. Results: After the identification of a suitable solid support for the study, the execution of the systematic comparison of both approaches showed that conventional and ultrasound-assisted syntheses yielded equivalent results with analogous composition of the raw products. Further, both approaches produced the affibody in good isolated yields of >20% when applying the same optimal reagent excesses and coupling times for the conjugation of each amino acid. This indicates that, under optimal reaction conditions, the choice of solid support used has a much stronger influence on the outcome of the preparation of Z_EGFR:1907 than the application of ultrasound, which did not further improve the synthesis results. Conclusions: Therefore, for the chemical synthesis of affibodies, great attention should be paid to the choice of a suitable solid support, enabling this highly interesting class of biomolecules to be obtained in good yields and to bring them more into the focus of radiopharmaceutical research. Full article

Human epidermal growth factor receptor 2 (HER2) is a major prognostic and predictive marker overexpressed in 15–20% of breast cancers. The diagnostic reference standard for selecting patients for HER2-targeted therapy is based on the analysis of tumor biopsies. Previously patients were defined as HER2-positive or -negative; however, with the approval of novel treatment options, specifically the antibody–drug conjugate trastuzumab deruxtecan, many breast cancer patients with tumors expressing low levels of HER2 have become eligible for HER2-targeted therapy. Such patients will need to be reliably identified by suitable diagnostic methods. Biopsy-based diagnostics are invasive, and repeat biopsies are not always feasible. They cannot visualize the heterogeneity of HER2 expression, leading to a substantial number of misdiagnosed patients. An alternative and highly accurate diagnostic method is molecular imaging with radiotracers. In the case of HER2, various studies demonstrate the clinical utility and feasibility of such approaches. Radiotracers based on Affibody^® molecules, small, engineered affinity proteins with a size of ~6.5 kDa, are clinically validated molecules with favorable characteristics for imaging. In this article, we summarize the HER2-targeted therapeutic landscape, describe our experience with imaging diagnostics for HER2, and review the currently available clinical data on HER2-Affibody-based molecular imaging as a novel diagnostic tool in breast cancer and beyond. Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6–7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody–IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody–IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody–IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function. Full article

Background: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. Methods: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. Results: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. Discussion: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors. Full article