Therapeutic affinity proteins offer a targeted mode of action due to their typically high affinity and specificity for disease-associated molecules. In cancer therapy, such target molecules are often overexpressed receptors on tumor cells. However, their presence in healthy tissues can lead to on-target, off-tumor toxicity, necessitating strategies to enhance tumor selectivity. Here, we present an affibody-based prodrug concept that exploits tumor-associated proteases for selective activation. As proof of concept, we designed, produced, and characterized HER2-specific prodrug candidates, each incorporating a distinct protease substrate for selective activation by tumor-associated proteases. Their activation by corresponding proteases and subsequent HER2 binding were assessed. The most promising prodrug candidate was conjugated to the cytotoxic agent DM1 and evaluated for cytotoxicity in HER2-positive cancer cells. The results demonstrated potent, HER2-dependent cell killing, with markedly reduced cytotoxicity in the absence of prodrug activation. These findings support the feasibility of affibody-based prodrugs as a strategy to enhance tumor selectivity and minimize off-tumor toxicity in targeted cancer therapy.

Overactive epidermal growth factor receptor (EGFR) signaling is often involved in driving different types of carcinomas. It is a well-studied target for targeted therapies, with both monoclonal antibodies and kinase inhibitors available for clinical use. Even though these drugs show a clinical benefit, most patients develop resistance over time. The development of new therapeutic modalities is therefore highly motivated. Herein, we describe a new type of drug candidate targeting EGFR, a so-called affibody-based drug conjugate. It consists of an EGFR-targeting affibody molecule, ZEGFR, expressed as a fusion to an albumin-binding domain for half-life extension, and coupled with the potent cytotoxic drug DM1 via a maleimidocaproyl linker. The resulting drug conjugate ZEGFR-ABD-mcDM1, showed strong binding to recombinant EGFR and EGFR-expressing cells. It was found to be highly potent in killing EGFR-expressing A431 cells with an IC50of 3.4 nM. In vivo, it showed moderate uptake in A431-derived xenografts with high EGFR expression. Collectively, the results from this study, demonstrate a potent and EGFR-specific drug candidate that holds promise for further development.

In vitro methods for developing binding domains have been well-established for many years, owing to the cost-efficient synthesis of DNA and high-throughput selection and screening technologies. However, generating high-affinity binding domains often requires the development of focused maturation libraries for a second selection, which typically demands a detailed understanding of the binding surfaces from the initial selection, a process that can be time-consuming. In this study, we accelerated this process by using deep sequencing data from the first selection to guide the design of the maturation library. Additionally, we employed a high-throughput screening system using flow cytometry based on Escherichia coli display to identify conditional binding domains from the selection output. This approach enabled the development of a high-affinity binder targeting the cancer biomarker HER3, with a binding affinity of 3.3 nM at extracellular pH 7.4, 100 times higher than the first-generation binding domain. Notably, the binding domain features a pH-dependent release mechanism, enabling rapid release in slightly acidic environments (pH ≈6), which resemble endosomal conditions. When conjugated to the cytotoxin mertansine (DM1), the binding domain demonstrated specific cytotoxic activity against HER3-expressing cell lines, with an IC50 of 2–5 nM. The presented approach enables the efficient development of conditional binding domains which hold promise for therapeutic applications.

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.

BACKGROUND: Heterozygous loss-of-function mutations in the gene encoding progranulin (GRN) are causative in around 5-10% of frontotemporal dementia (FTD) cases. These mutations lead to a more than 50% reduction of progranulin levels in the plasma and cerebrospinal fluid of mutation carriers compared to healthy controls. Increasing the progranulin levels in FTD-GRN patients via inhibition of sortilin-mediated progranulin degradation has shown promise as a therapeutic strategy, as exemplified by Alector's latozinemab, currently in phase 3 clinical trials. Here we detail the development of an anti-sortilin affibody-based miniprotein as a non-immunoglobulin alternative, offering potential advantages due to its small size and cost-effective production. METHOD: Sortilin-binding affibodies were selected by phage display and subsequently genetically fused to short peptides derived from the progranulin C-terminus. The resulting miniproteins were characterized in terms of affinity, structure, stability, and their ability to increase extracellular progranulin levels in a clearance assay using progranulin-secreting, sortilin-expressing U-251 MG cells. RESULT: A set of moderate-affinity sortilin-binding affibodies were obtained from phage display selections. Following genetic fusion with short peptides derived from the progranulin C-terminus and optimization of the fusion constructs, a lead candidate with 185 pM affinity for sortilin was obtained. The affibody-peptide fusion, but not its parental affibody or peptide, was capable of elevating extracellular progranulin levels in vitro with similar potency as latozinemab. CONCLUSION: A sortilin-binding affibody-based miniprotein was developed and optimized, performing on par with latozinemab in in vitro functional studies. Affibody-based miniproteins provide a promising alternative to immunoglobulins in cases such as the present, where Fc functions are undesirable, and long-term high-dose antibody treatments risk becoming prohibitively expensive.

Proteases play a crucial role in biological functions such as tumor progression and tissue homeostasis. Recently, protease-activated prodrugs have gained attention for their potential to enhance selectivity in tumor-targeted therapies. In this study, we report the engineering of substrate sequences for matriptase, a protease overexpressed in tumors and previously explored for prodrug activation in vivo. A peptide library containing millions of potential substrates was displayed on Escherichia coli, and flow cytometric sorting was used to isolate improved substrates based on cleavage efficiency. Hits were ranked by flow cytometry, and the top substrates exhibited kcat /KM values over 40-fold higher than previously reported sequences. These substrates were further evaluated in an antibody-prodrug format, demonstrating exceptional activation. The matriptase substrates hold broad potential for applications such as cleavable linkers in next-generation antibody prodrugs. Furthermore, the developed bacterial display platform shows promise for discovering substrates of other proteases.

Background

Lung diseases such as idiopathic pulmonary fibrosis and acute respiratory distress syndrome (ARDS) are associated with significant morbidity and mortality, with limited treatment options. Platelet-derived growth factor receptor beta (PDGFR beta) signaling pathway is a key driver of fibrogenesis in different organs. In the lungs, pericytes have a high PDGFR beta expression, and their role as immune regulators and progenitors of myofibroblasts is increasingly recognized. Non-invasive techniques to assess active lung tissue remodeling are needed to improve disease monitoring and treatment evaluation.

This study aimed to evaluate [18F]TZ-Z09591, targeting PDGFR beta, for imaging pulmonary injuries in human biopsies, and in vivo in animal models of lung injury.Results[18F]TZ-Z09591 demonstrated high and specific binding to PDGFR beta-expressing cells. Autoradiography confirmed tracer uptake in lung injuries, including fibrotic foci, from human, rat, and pig lung tissues. In vivo positron emission tomography (PET) imaging of bleomycin-induced lung fibrosis in rats and an ARDS pig model showed significantly increased uptake in diseased lung segments compared to controls, especially in pulmonary injuries with collagen deposition, despite moderate background uptake.

Conclusions

This study demonstrated that [18F]TZ-Z09591 can assess PDGFR beta expression in pulmonary injuries, supporting its potential for non-invasive assessment of lung tissue remodeling. PET imaging targeting PDGFR beta could improve disease monitoring, and provide new insights into pulmonary fibrosis progression.

This review outlines the historical development and versatile applications of one of the most well-studied bacterial proteins, namely the immunoglobulin (Ig)-binding staphylococcal protein A (SpA) of Staphylococcus aureus. Each segment of the SpA operon, from the 5’ promoter region and signal peptide to the 3’ cell wall anchoring region, has been exploited for various innovative applications in areas such as immunology and biotechnology. We provide an overview of selected applications and concepts that have had a significant impact on life science research, and some that have also led to significant commercial implications. In the 1980s, the SpA promoter and signal sequence were utilized in Escherichia coli for recombinant production of various proteins, yielding product secretion to the culture medium and thereby simplifying product recovery. The five homologous Ig-binding domains of SpA gained tremendous interest in the late 1980s, largely due to the rise of monoclonal antibodies (mAbs) for therapeutic use, prompting a growing demand for effective affinity ligands to facilitate their purification. Over the years, these Ig-binding domains have been extensively investigated and re-engineered to bind proteins other than antibodies, leading in the mid-1990s to the development of the affibody affinity protein technology. Today, affibody molecules are being investigated in late-stage clinical trials as potential protein therapeutics for various indications. Finally, the cell wall anchoring regions of SpA inspired the development of a surface display system for Staphylococcus carnosus, which has emerged as a technology platform in combinatorial protein engineering for work with large peptide, antibody and affibody libraries.

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-PGRNC15*) 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 EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* 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.

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., >10⁷ variants) and is described here.