A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents, and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue, in both pancreas and in islet grafts. The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas, but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here, we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells, and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method, new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity, suitable stability and biodistribution, with negligible toxicity to islets, qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf>, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell–deficient I-A^g7 major histocompatibility complex class II–expressing recipient mice. The mice were examined longitudinally by [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> in the paws was analyzed and expressed as SUV<inf>mean</inf>. Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5–7 d after induction of arthritis. The uptake of [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf>. Conclusion: The uptake of [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [⁶⁸Ga]Ga-DOTA-Z<inf>CAM241</inf> is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.

Non-invasive assessment of fibrogenic activity, rather than fibrotic scars, could significantly improve the management of fibrotic diseases and the development of anti-fibrotic drugs. This study explores the potential of an Affibody molecule (Z09591) labeled with the Al(18)F-restrained complexing agent (RESCA) method as a tracer for the non-invasive detection of fibrogenic cells. Z09591 was functionalized with the RESCA chelator for direct labeling with [18F]AlF. 18 F]AlF. In vivo positron emission tomography/magnetic resonance imaging scans on U-87 tumor-bearing mice exhibited high selectivity of the resulting radiotracer, [18F]AlF-RESCA-Z09591, 18 F]AlF-RESCA-Z09591, for platelet-derived growth factor receptor b (PDGFRb), b ), with minimal non-specific background uptake. Evaluation in a mouse model with carbon tetrachloride-induced fibrotic liver followed by a disease regression phase, revealed the radiotracer's high affinity and specificity for fibrogenic cells in fibrotic livers (standardized uptake value [SUV] 0.43 +/- 0.05), with uptake decreasing during recovery (SUV 0.29 +/- 0.03) (p p < 0.0001). [18F]AlF-RESCA-Z09591 18 F]AlF-RESCA-Z09591 accurately detects PDGFRb, b, offering noninvasive assessment of fibrogenic cells and promising applications in precise liver fibrogenesis diagnosis, potentially contributing significantly to anti-fibrotic drug development.

Background: Platelet-derived growth factor receptor beta (PDGFRβ) is a receptor overexpressed on activated hepatic stellate cells (aHSCs). Positron emission tomography (PET) imaging of PDGFRβ could potentially allow the quantification of fibrogenesis in fibrotic livers. This study aims to evaluate a fluorine-18 radiolabeled Affibody molecule ([18F]TZ-Z09591) as a PET tracer for imaging liver fibrogenesis. Results: In vitro specificity studies demonstrated that the trans-Cyclooctenes (TCO) conjugated Z09591 Affibody molecule had a picomolar affinity for human PDGFRβ. Biodistribution performed on healthy rats showed rapid clearance of [18F]TZ-Z09591 through the kidneys and low liver background uptake. Autoradiography (ARG) studies on fibrotic livers from mice or humans correlated with histopathology results. Ex vivo biodistribution and ARG revealed that [18F]TZ-Z09591 binding in the liver was increased in fibrotic livers (p = 0.02) and corresponded to binding in fibrotic scars. Conclusions: Our study highlights [18F]TZ-Z09591 as a specific tracer for fibrogenic cells in the fibrotic liver, thus offering the potential to assess fibrogenesis clearly. Graphical abstract: [Figure not available: see fulltext.]

Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.

Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a K-d of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant Z(CD69:4) had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule Z(CD69:4) is suitable for further development for imaging of activated immune cells.

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.