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Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for selection via multiple display methods. This protocol describes selection from affibody libraries by Escherichia coli cell surface display. With this method, high-diversity libraries of 10 11 can be displayed on the cell surface. The method involves two steps for selection of binders from high-diversity libraries: magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is used first to enrich the library in target-binding clones and to decrease diversity to a size that can be effectively screened and sorted in the flow cytometer in a reasonable time (typically <10 7 cells). The protocol is based on methodology using an AIDA-I autotransporter for display on the outer membrane, but the general procedures can also be adjusted and used for other types of autotransporters or alternative E. coli display methods., (© 2024 Cold Spring Harbor Laboratory Press.)

Affibody molecules are small (6-kDa) affinity proteins folded in a three-helical bundle and generated by directed evolution for specific binding to various target molecules. The most advanced affibody molecules are currently tested in the clinic, and data from more than 300 subjects show excellent activity and safety profiles. The generation of affibody molecules against a particular target starts with the generation of an affibody library, which can then be used for panning using multiple methods and selection systems. This protocol describes the molecular cloning of DNA-encoded affibody libraries to a display vector of choice, for either phage, Escherichia coli , or Staphylococcus carnosus display. The DNA library can come from different sources, such as error-prone polymerase chain reaction (PCR), molecular shuffling of mutations from previous selections, or, more commonly, from DNA synthesis using various methods. Restriction enzyme-based subcloning is the most common strategy for affibody libraries of higher diversity (e.g., >10 7 variants) and is described here., (© 2024 Cold Spring Harbor Laboratory Press.)

Affibody molecules are small, robust, and versatile affinity proteins currently being explored for therapeutic, diagnostic, and biotechnological applications. Surface-exposed residues on the affibody scaffold are randomized to create large affibody libraries from which novel binding specificities to virtually any protein target can be generated using combinatorial protein engineering. Affibody molecules have the potential to complement-or even surpass-current antibody-based technologies, exhibiting multiple desirable properties, such as high stability, affinity, and specificity, efficient tissue penetration, and straightforward modular extension of functional domains. It has been shown in both preclinical and clinical studies that affibody molecules are safe, efficacious, and valuable alternatives to antibodies for specific targeting in the context of in vivo diagnostics and therapy. Here, we provide a general background of affibody molecules, give examples of reported applications, and briefly summarize the methodology for affibody generation., (© 2024 Cold Spring Harbor Laboratory Press.)

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library. This is then followed by amplification of the library, which can then be used for biopanning using multiple methods. This protocol describes amplification of affibody libraries, followed by biopanning using phage display and analysis of the selection output. The general procedure is mainly for selection of first-generation affibody molecules from large naive (unbiased) libraries, typically yielding affibody hits with affinities in the low nanomolar range. For selection from affinity maturation libraries with the aim of isolating variants of even higher affinities, the procedure is similar, but parameters such as target concentration and washing are adjusted to achieve the proper stringency., (© 2024 Cold Spring Harbor Laboratory Press.)

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed., (© 2024 Cold Spring Harbor Laboratory Press.)

Background: In recent years, the interest in Al[ 18 F]F as a labeling agent for Positron Emission Tomography (PET) radiotracers has risen, as it allows for fast and efficient fluorine-18 labeling by harnessing chelation chemistry. The introduction of Restrained Complexing Agent (RESCA) as a chelator has also shown that chelator-based radiolabeling reactions can be performed in mild conditions, making the radiolabeling process attractively more facile than most conventional radiofluorination methods. The aim of the study was to establish optimized conditions for Al[ 18 F]F labeling of Affibody molecules using RESCA as a complexing agent, using Z 09591 and Z 0185 , two Affibody proteins targeting PDGFRβ and TNFα, respectively, as model compounds., Results: The Al[ 18 F]F labeling of RESCA-conjugated Z 09591 was tested at different temperatures (rt to 60 °C) and with varying reaction times (12 to 60 min), and optimal conditions were then implemented on RESCA-Z 0185 . The optimized synthesis method was: 1.5-2.5 GBq of cyclotron produced fluorine-18 were trapped on a QMA cartridge and eluted with saline solution to react with 12 nmol of AlCl 3 and form Al[ 18 F]F. The respective RESCA-conjugated Affibody molecule (14 nmol) in NaOAc solution was added to the Al[ 18 F]F solution and left to react at 60 °C for 12 min. The mixture was purified on a NAP5 size exclusion column and then analyzed by HPLC. The entire process took approximately 35 min, was highly reproducible, indicating the efficiency and reliability of the method. The labeled compounds demonstrated retained biological function for their respective targets after purification., Conclusions: We present a general and optimized method for Al[ 18 F]F labeling of RESCA-conjugated Affibody molecules, which can be widely applied to this class of peptide-based imaging agents. Moreover, radiochemical yields were improved when the labeling was conducted at 37 °C or above. In vitro and in vivo assessment of the respective tracers was promising, showing retained binding capacity as well as moderate defluorination, which is usually regarded as a potential downside for RESCA-conjugated tracers., (© 2024. The Author(s).)

Bacterial surface display in combination with fluorescence-activated cell sorting is a versatile and robust system and an interesting alternative approach to phage display for the generation of therapeutic affinity proteins. The system enables real-time monitoring and sorting of cell populations, which presents unique possibilities for drug development. It has been used to develop several affibody molecules currently being evaluated preclinically for the treatment and diagnosis of, for example, cancer and neurodegenerative diseases. Additionally, it can be implemented in other areas of drug design, such as for mapping epitopes and evolving enzyme specificities., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody binding. Hence, these variants contains molecular "on/off" switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications., Competing Interests: Conflict of interest S. H. and M. J. have filed a patent application regarding use of the domain as a purification tool., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The outcome of clinical trials evaluating drugs targeting the human epidermal growth factor receptor 3 (HER3) has been poor, with primary concerns related to lack of efficacy. HER3 is considered a difficult target since its overexpression on tumors is relatively low and there is normal expression in many different organs. However, a significant number of patients across different cancer indications have overexpression of HER3 and the development of novel modalities targeting HER3 is therefore warranted. Here, we have investigated the properties of affibody-based drug conjugates targeting HER3. The HER3-targeting affibody molecule Z HER3 was fused in a mono- and bivalent format to an engineered albumin-binding domain (ABD) for in vivo half-life extension and was coupled to the cytotoxic drug DM1 via a non-cleavable maleimidocaproyl (mc) linker. In vivo , a moderate uptake was observed for [ 99m Tc]Tc-labeled Z HER3 -ABD-Z HER3 -mcDM1 in HER3 expressing BxPC3 tumors (3.5 ± 0.3%IA/g) at 24 h after injection, and clearance was predominately renal-mediated. Treatment of mice with BxPC3 human pancreatic cancer xenografts showed that a combination of Z HER3 -ABD-Z HER3 -mcDM1 and its cytostatic analog Z HER3 -ABD-Z HER3 was efficacious and superior to treatment with only Z HER3 -ABD-Z HER3 , providing tumor growth inhibition and longer median survival (90 d) in comparison to monotherapy (68 d) and vehicle control (49 d). Z HER3 -ABD-Z HER3 -mcDM1 was found to be a potent drug conjugate for the treatment of HER3-expressing tumors in mice., Competing Interests: The authors declare the following competing financial interest(s): Anna Orlova, Stefan Stahl, John Lofblom, Torbjorn Graslund, and Anzhelika Vorobyeva are founders and own shares in Zytox Therapeutics AB., (© 2024 The Authors. Published by American Chemical Society.)

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro . By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRN C 15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC 50 value of 1.3 nM. Our results introduce A3-PGRN C 15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units., Competing Interests: HL, P-ÅN, SS and JL are shareholders of Amylonix AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ek, Nilvebrant, Nygren, Ståhl, Lindberg and Löfblom.)

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 [ 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, [ 18 F]AlF-RESCA-Z09591, for platelet-derived growth factor receptor β (PDGFRβ), 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  < 0.0001). [ 18 F]AlF-RESCA-Z09591 accurately detects PDGFRβ, offering non-invasive assessment of fibrogenic cells and promising applications in precise liver fibrogenesis diagnosis, potentially contributing significantly to anti-fibrotic drug development., Competing Interests: B.M. and O.E. are employees of Antaros Medical AB. F.Y.F. is an employee of Affibody AB. O.K. and O.E. are shareholders of Antaros Tracer AB., (© 2024 The Authors.)

Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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, [ 68 Ga]Ga-DOTA-Z CAM241 , 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 [ 68 Ga]Ga-DOTA-Z CAM241 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 [ 68 Ga]Ga-DOTA-Z CAM241 in the paws was analyzed and expressed as SUV mean 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 [ 68 Ga]Ga-DOTA-Z CAM241 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 [ 68 Ga]Ga-DOTA-Z CAM241 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 [ 68 Ga]Ga-DOTA-Z CAM241 Conclusion: The uptake of [ 68 Ga]Ga-DOTA-Z CAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [ 68 Ga]Ga-DOTA-Z CAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

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 Z DGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [ 18 F]Z DGCR2: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 [ 18 F]Z DGCR2:AM106 was also assessed in healthy rats and pigs., Results: [ 18 F]Z DGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [ 18 F]Z DGCR2: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 [ 18 F]Z DGCR2: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 [ 18 F]Z DGCR2: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 [ 18 F]Z DGCR2: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., (© 2023. The Author(s).)

Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering-based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi-rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

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 ([ 18 F]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 [ 18 F]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 [ 18 F]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., (© 2023. Springer Nature Switzerland AG.)

The blood-brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engineered shuttle proteins facilitates ranking and the selection of promising candidates during development. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. My Hedhammar reports a relationship with Spiber Technologies AB that includes: equity or stocks., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recognition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide technology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 10 9 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody Z EGFR:2377 ) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease-dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for administration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (Z B05 ). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of Z B05 . In this study we evaluate a novel affibody-based prodrug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted therapeutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SS is a minority shareholder of Affibody AB. The other authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Conditional activation of engineered affinity proteins by proteolytic processing is an interesting approach for a wide range of applications. We have generated an anti-idiotypic masking domain with specificity for the binding surface of an EGFR-targeting affibody molecule using an in-house developed staphylococcal display method. The masking domain could specifically abrogate EGFR-binding on cancer cells when fused to the EGFR-targeting affibody molecule via a linker comprising a protease cleavage site. EGFR-binding was restored by proteolytic cleavage of the linker region resulting in release of the masking domain. A saturation mutagenesis study provided detailed information on the interaction between the EGFR-targeting affibody molecule and the masking domain. Introducing an anti-idiotypic masking affibody domain is a viable approach for blocking EGFR-binding and allows for conditional activation by proteolytic processing. The results warrant further studies evaluating the therapeutic and diagnostic applicability both in vitro and in vivo., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SS is a minority shareholder of Affibody AB. The other authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.

This review describes the principles for generation of affibody molecules using bacterial display on the Gram-negative Escherichia coli and the Gram-positive Staphylococcus carnosus, respectively. Affibody molecules are small and robust alternative scaffold proteins that have been explored for therapeutic, diagnostic, and biotechnological applications. They typically exhibit high-stability, affinity, and specificity with high modularity of functional domains. Due to the small size of the scaffold, affibody molecules are rapidly excreted through renal filtration and can efficiently extravasate from blood and penetrate tissues. Preclinical and clinical studies have demonstrated that affibody molecules are promising and safe complements to antibodies for in vivo diagnostic imaging and therapy. Sorting of affibody libraries displayed on bacteria using fluorescence-activated cell sorting is an effective and straightforward methodology and has been used successfully to generate novel affibody molecules with high affinity for a diverse range of molecular targets., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer's disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood-brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125 I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [ 125 I]I-Z5 and [ 125 I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [ 125 I]I-scFv8D3-Z5 and [ 125 I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [ 125 I]I-Z1 and [ 125 I]I-Z5 was low, while [ 125 I]I-scFv8D3-Z1 and [ 125 I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [ 125 I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [ 125 I]I-scFv8D3-Z5 and [ 125 I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits., (© 2022. The Author(s).)

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, Z HER3 -ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. Z HER3 -ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (K D 6 nM), and an even stronger affinity (K D 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC 50 value of 7 nM. Evaluation of a radiolabeled version, [ 99m Tc]Tc-Z HER3 -ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that Z HER3 -ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

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., (© 2021. The Author(s).)

HER3 (human epidermal growth factor receptor type 3) is a challenging target for diagnostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89 Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab) 2 -fragment labeled with 89 Zr and 68 Ga, and the 68 Ga-labeled affibody molecule [ 68 Ga]Ga-Z HER3 . The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribution was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [ 68 Ga]Ga-DFO-seribantumab-F(ab') 2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [ 89 Zr]Zr-DFO-seribantumab and [ 89 Zr]Zr-DFO-seribantumab-F(ab') 2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [ 68 Ga]Ga-Z HER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.

Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [ 68 Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68 Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66 Ga (T 1/2 = 9.49 h, β + = 56.5%) would permit imaging with higher contrast. 66 Ga was produced by the 66 Zn(p,n) 66 Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66 Ga with preserved binding specificity in vitro and in vivo. At 24 h after injection, [ 66 Ga]Ga-DFO-ZEGFR:2377 provided 3.9-fold higher tumor-to-blood ratio and 2.3-fold higher tumor-to-liver ratio than [ 68 Ga]Ga-DFO-ZEGFR:2377 at 3 h after injection. At the same time point, [ 66 Ga]Ga-DFO-ZEGFR:2377 provided 1.8-fold higher tumor-to-blood ratio, 3-fold higher tumor-to-liver ratio, 1.9-fold higher tumor-to-muscle ratio and 2.3-fold higher tumor-to-bone ratio than [ 89 Zr]Zr-DFO-ZEGFR:2377. Biodistribution data were confirmed by whole body PET combined with magnetic resonance imaging (PET/MRI). The use of the positron emitter 66 Ga for labelling of DFO-ZEGFR:2377 permits PET imaging of EGFR expression at 24 h after injection and improves imaging contrast.

The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 °C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.

Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [ 68 Ga]Ga-(HE) 3 -Z 08698 -NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.

Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post-mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We describe here a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights into the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood-brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (Z SYM73 ) via binding to the transferrin receptor (TfR). Z SYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer's disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule Z SYM73 . Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE) 3 -Z HER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE) 3 -Z HER3 -X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [ 57 Co]Co (surrogate for 55 Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA 3 h pi, [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [ 57 Co]Co-chelator complex influenced the uptake in tumors and normal tissue. [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA improved imaging contrast compared to early-time-point imaging with [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA.