Alternative Protein Scaffolds Research Articles

Enzymatic CO2 reduction catalyzed by natural and artificial Metalloenzymes

The continuously increasing level of atmospheric CO2 in the atmosphere has led to global warming. Converting CO2 into other carbon compounds could mitigate its atmospheric levels and produce valuable products, as CO2 also serves as a plentiful and inexpensive carbon feedstock. However, the inert nature of CO2 poses a major challenge for its reduction. To meet the challenge, nature has evolved metalloenzymes using transition metal ions like Fe, Ni, Mo, and W, as well as electron-transfer partners for their functions. Mimicking these enzymes, artificial metalloenzymes (ArMs) have been designed using alternative protein scaffolds and various metallocofactors like Ni, Co, Re, Rh, and FeS clusters. Both the catalytic efficiency and the scope of CO2-reduction product of these ArMs have been improved over the past decade. This review first focuses on the natural metalloenzymes that directly reduce CO2 by discussing their structures and active sites, as well as the proposed reaction mechanisms. It then introduces the common strategies for electrochemical, photochemical, or photoelectrochemical utilization of these native enzymes for CO2 reduction and highlights the most recent advancements from the past five years. We also summarize principles of protein design for bio-inspired ArMs, comparing them with native enzymatic systems and outlining challenges and opportunities in enzymatic CO2 reduction.

Secondary structure changes as the potential H2 sensing mechanism of group D [FeFe]-hydrogenases.

[FeFe]-hydrogenases function as both H2 catalysts and sensors. While catalysis is well investigated, details regarding the H2 sensing mechanism are limited. Here, we relate protein structure changes to H2 sensing, similar to light-driven bio-sensors. Our results highlight how identical cofactors incorporated in alternative protein scaffolds serve different functions in nature.

Inhalable Nanofitin demonstrates high neutralization of SARS-CoV-2 virus via direct application in respiratory tract

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in invitro and invivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

Bacterial Cell Display for Selection of Affibody Molecules.

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.

TCR mimic compounds for pHLA targeting with high potency modalities in oncology.

pHLA complexes represent the largest class of cell surface markers on cancer cells, making them attractive for targeted cancer therapies. Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology.

Clinical possibilities of HER2-positive breast cancer diagnosis using alternative scaffold proteins

HER2-positive breast cancer occurs in 15–20% of breast cancer patients and is associated primarily with a poor prognosis of the disease and the need for highly specific targeted therapy. Despite the clinical importance of determining HER2/neu, traditional diagnostic methods have their disadvantages and require the study of new additional research techniques.The information presented in this review makes it possible to consider current trends in the radionuclide diagnosis of HER2-positive breast cancer using the latest class of alternative scaffold proteins and to consider various aspects of their use in clinical practice.

ПЕРВЫЙ ОПЫТ КЛИНИЧЕСКОГО ИСПОЛЬЗОВАНИЯ ПРЕПАРАТА «99mTc-DARPinG3» ДЛЯ РАДИОНУКЛИДНОЙ ДИАГНОСТИКИ РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ С ГИПЕРЭКСПРЕССИЕЙ HER2/neu

Purpose: To study the possibility of clinical use of the radiopharmaceutical 99mТс – DARPinG3 for the diagnosis of breast cancer with HER2 / neu overexpression in humans. Material and methods: The clinical study was registered with ClinicalTrials.gov Identifier: NCT04277338 and approved by the Bioethical Committee of the Research Institute of Oncology of the Tomsk National Research Medical Center. The study included 9 breast cancer patients (T1-4N0-2M0-1) before systemic treatment: 5 – with HER2/neu overexpression; 4 – with negative expression. In all cases, a morphological and immunohistochemical study of the tumor biopsy material was carried out. The dosage of the DARPinG3 protein was 1000 μg; labeling was carried out according to the tricarbonyl technique. WholeBody scintigraphy and single-photon emission computed tomography were performed on an E.CAM 180 gamma camera from Siemens (Germany) at 2, 4, 6 and 24 hours after injection. Results: The first clinical studies of 99mTc – DARPinG3 at a dosage of 1000 µg demonstrated the safety and the absence of toxic effects on patients with breast cancer. The radiopharmaceutical demonstrated rapid elimination from the bloodstream and an specific effective dose (0.011 ± 0.001 mSv / MBq), comparable to results of other representatives of alternative scaffold proteins labeled with various isotopes. The highest accumulation of the labeled protein was observed in patients with HER2-positive breast tumors at 2 and 4 hours after injections (p <0.05, Mann‒Whitney test). Conclusion: The obtained results indicate that 99mTc-DARPinG3 is safe for clinical use in human and can be considered as a new additional method for diagnosing HER2-positive breast tumors.

Comparative analysis of the use of technetium-99m labeled recombinant target molecules at doses of 1000 and 2000 μg for radionuclide diagnostics of HER2-positive breast cancer

Materials and methods. The study included 18 patients with breast cancer (T1–4N0–2M0) before systemic treatment: 10 — with HER2/neu overexpression; 8 — HER2/neu negative. In all cases, morphological, immunohistochemical studies, as well as FISH analysis of the primary tumor were performed. All patients underwent radionuclide studies (planar scintigraphy and SPECT of the chest) at 2, 4, 6 and 24 hours after injection of the «99mTc-DARPinG3» in dosages of 1000 and 2000 μg. Results. The half-life of the labeled protein from the blood was 3.5 hours for a dosage of 1000 μg; 3.8 h — for 2000 mcg. The organ with the highest absorption of «99mTc-DARPinG3» was the kidneys, regardless of the protein dose (0.10±0.02 and 0.10±0.03 mGy, respectively). The effective dose for 1000 μg was 0.011±0.001 mGy; for 2000 μg — 0.012±0.006. The liver absorption dose was significantly higher with 1000 μg of protein compared to 2000 μg (p<0.005, Mann–Whitney U test). The tumor/background ratio was significantly higher at 2 and 4 hours after administration of the «99mTc-DARPinG3» in patients with HER2-positive tumors in 1000 and 2000 μg doses of protein (p<0.005, Mann–Whitney U test). Conclusion. Clinical studies of the «99mTc-DARPinG3» in dosages of 1000 and 2000 μg demonstrated rapid elimination from the bloodstream and effective doses comparable to those obtained in the study with others alternative scaffold proteins labeled with various isotopes. An essential point of this work is the positive correlation of the protein dose with the accumulation of the compound in the liver, which is important component in diagnostic and allows to detect tumor sites in liver.

The Evolution of Targeted Radionuclide Diagnosis of HER2-Positive Breast Cancer.

This review examines the evolution of the radionuclide diagnosis of HER2-positive breast cancer using various compounds as a targeting module in clinical practice: from full-length antibodies to a new group of small synthetic proteins called alternative scaffold proteins. This topic is of especial relevance today in view of the problems attendant to the detection of breast cancer with HER2/neu overexpression, which, in most cases, introduce errors in the treatment of patients. The results of clinical studies of radiopharmaceuticals based on affibody molecules, ADAPTs, and DARPins for SPECT and PET have demonstrated good tolerability of the compounds, their rapid excretion from the body, and the possibility to differentiate tumor sites depending on the HER2/neu status. This indicates that targeted radionuclide diagnosis holds promise and the need to continue research in this direction.

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens.

Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.

Determination of the most informative prognostic parameters for assessing the status of the epidermal growth factor receptor Her2/neu in the primary tumor in breast cancer patients using the targeted radiopharmaceutical «99mTc-ADAPT6»

The aim of the study was to identify the most informative prognostic parameters that allow assessing the status of the epidermal growth factor receptor Her2/neu in the primary tumor in breast cancer patients using the radiopharmaceutical «99mTc-ADAPT6».
 Material and methods. The study included 15 patients with breast cancer (T1-4N0-2M0) before the system therapy. Eight patients were found to overexpress Her2/neu in the primary breast tumor, in 7 cases the expression of the marker was not diagnosed. Morphological and immunohistochemical studies were performed in all cases. All patients are undergoing a radionuclide study using the radiopharmaceutical «99mTc-ADAPT6» at a dosage of protein 500 μg.
 Results. Patients with Her2-positive breast tumors showed a significantly higher accumulation of «99mTc-ADAPT6» in primary tumor (p<0.05, Mann-Whitney U test), as well as high tumor/background, tumor/latissimus dorsi muscle and tumor/liver as compared with patients with negative status of Her2/neu (p<0.005, Mann-Whitney U test). Based on the analysis performed using the logistic regression method, there was a mathematical model for predicting the Her2/neu status in primary tumor of breast cancer patients (specificity and sensitivity 100%; p=0.0002)
 Conclusion. The results of the study make it possible to consider the radiopharmaceutical «99mTc-ADAPT6» as an additional promising agent for determining the Her2/neu status in breast cancer patients.
 Key words: breast cancer, radionuclide diagnostics, alternative scaffold proteins, ADAPT6, Her2 / neu

Comparative Analysis of the Clinical Use of 99mTechnetium-Labeled Recombinant Target Molecules in Different Dosages for the Radionuclide Diagnosis of Her2-Positive Breast Cancer

Background. The chief goal in assessing the Her2/neu status in clinical practice is to identify indications for targeted therapy. The main methods for determining the Her2/neu status are an immunohistochemical method and fluorescence in situ hybridization (FISH); however, despite their widespread use, they have a number of significant disadvantages. Over the past few years, radionuclide diagnosis using a new class of alternative scaffold proteins that meet all the requirements for optimal radionuclide delivery to tumor cells has become widespread. Objective: comparative analysis of the effectiveness of radionuclide imaging of Her2-positive breast cancer using 99m technetium-labeled recombinant molecules in different dosages. Material and methods. The investigation enrolled 22 patients with breast cancer (T1-4N0-2M0) before systemic therapy: 11 had Her2/neu overexpression; 11 had no marker expression. The patients’ mean age was 50.7 ± 2.3 years. Morphological and immunohistochemical studies were performed in all cases. FISHanalysis was carried out in the presence of the Her2/neu 2+ value. The agent was prepared immediately before using in dosages of 500 and 1000 µg, after which it was slowly administered intravenously to the patient. Whole-body scintigraphy and chest single-photon emission computed tomography were conducted at 2, 4, 6 and 24 hours after administration. Results. The radiochemical yield and radiochemical purity values were 77 ± 9% and 99 ± 1%, respectively. The activity of the agent immediately before administration was 416 ± 135 MBq for the 500 µg group and 349 ± 133 MBq for 1000 µg group. Analysis of the findings indicated that the higher uptake of the agent by organs without tumor lesion was observed in the kidneys, liver, and lungs. The highest renal absorption of the agent was observed in both study groups (0.135 ± 0.042 and 0.191 ± 0.047 mGy, respectively). The effective dose was 0.009 ± 0.002 mGy for the 500 µg group and 0.010 ± 0.003 mGy for the 1000 µg group. The better distribution between the tumors with Her2/neu positive and negative statuses was observed 2 hours after administration in the 500 µg group with the mean tumor/background value of 37 ± 19 for Her2-positive tumors, and 5 ± 2 for Her2-negative tumors (p < 0.001). Conclusion. The findings suggest that the test radiopharmaceutical agent at a dose of 500 µg can be considered as a new additional method to diagnose Her2-positive breast tumors.

Centyrin ligands for extrahepatic delivery of siRNA

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1.

Cost of Manufacturing for Recombinant Snakebite Antivenoms.

Snakebite envenoming is a neglected tropical disease that affects millions of people across the globe. It has been suggested that recombinant antivenoms based on mixtures of human monoclonal antibodies, which target key toxins of medically important snake venom, could present a promising avenue toward the reduction of morbidity and mortality of envenomated patients. However, since snakebite envenoming is a disease of poverty, it is pivotal that next-generation therapies are affordable to those most in need; this warrants analysis of the cost dynamics of recombinant antivenom manufacture. Therefore, we present, for the first time, a bottom-up analysis of the cost dynamics surrounding the production of future recombinant antivenoms based on available industry data. We unravel the potential impact that venom volume, abundance of medically relevant toxins in a venom, and the molecular weight of these toxins may have on the final product cost. Furthermore, we assess the roles that antibody molar mass, manufacturing and purification strategies, formulation, antibody efficacy, and potential cross-reactivity play in the complex cost dynamics of recombinant antivenom manufacture. Notably, according to our calculations, it appears that such next-generation antivenoms based on cocktails of monoclonal immunoglobulin Gs (IgGs) could be manufacturable at a comparable or lower cost to current plasma-derived antivenoms, which are priced at USD 13-1120 per treatment. We found that monovalent recombinant antivenoms based on IgGs could be manufactured for USD 20-225 per treatment, while more complex polyvalent recombinant antivenoms based on IgGs could be manufactured for USD 48-1354 per treatment. Finally, we investigated the prospective cost of manufacturing for recombinant antivenoms based on alternative protein scaffolds, such as DARPins and nanobodies, and highlight the potential utility of such scaffolds in the context of low-cost manufacturing. In conclusion, the development of recombinant antivenoms not only holds a promise for improving therapeutic parameters, such as safety and efficacy, but could possibly also lead to a more competetive cost of manufacture of antivenom products for patients worldwide.

Development and activities, including immunocomplex formation, of biparatopic antibodies and alternative scaffold proteins

Development and activities, including immunocomplex formation, of biparatopic antibodies and alternative scaffold proteins

High-Throughput Quantification of Surface Protein Internalization and Degradation.

Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.

Toxin Neutralization Using Alternative Binding Proteins.

Animal toxins present a major threat to human health worldwide, predominantly through snakebite envenomings, which are responsible for over 100,000 deaths each year. To date, the only available treatment against snakebite envenoming is plasma-derived antivenom. However, despite being key to limiting morbidity and mortality among snakebite victims, current antivenoms suffer from several drawbacks, such as immunogenicity and high cost of production. Consequently, avenues for improving envenoming therapy, such as the discovery of toxin-sequestering monoclonal antibodies against medically important target toxins through phage display selection, are being explored. However, alternative binding protein scaffolds that exhibit certain advantages compared to the well-known immunoglobulin G scaffold, including high stability under harsh conditions and low cost of production, may pose as possible low-cost alternatives to antibody-based therapeutics. There is now a plethora of alternative binding protein scaffolds, ranging from antibody derivatives (e.g., nanobodies), through rationally designed derivatives of other human proteins (e.g., DARPins), to derivatives of non-human proteins (e.g., affibodies), all exhibiting different biochemical and pharmacokinetic profiles. Undeniably, the high level of engineerability and potentially low cost of production, associated with many alternative protein scaffolds, present an exciting possibility for the future of snakebite therapeutics and merit thorough investigation. In this review, a comprehensive overview of the different types of binding protein scaffolds is provided together with a discussion on their relevance as potential modalities for use as next-generation antivenoms.

Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry.

Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.

Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display.

Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.