Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules.

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

Identification of fibronectin type III domain containing 3B as a potential prognostic and therapeutic target for pancreatic cancer: a preliminary analysis.

BACKGROUND: Fibronectin type III domain containing 3B (FNDC3B), a member of the fibronectin type III domain-containing protein family, has been indicated in various malignancies. However, the precise role of FNDC3B in the progression of pancreatic cancer (PC) still remains to be elucidated. METHODS: In this study, we integrated data from the National Center for Biotechnology Information, the Cancer Genome Atlas, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets to analyze FNDC3B expression and its association with various clinicopathological parameters. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, along with Gene Set Enrichment Analysis (GSEA), single sample Gene Set Enrichment Analysis (ssGSEA) and estimate analysis were recruited to delve into the biological function and immune infiltration based on FNDC3B expression. Additionally, the prognostic estimation was conducted using Cox analysis and Kaplan-Meier analysis. Subsequently, a nomogram was constructed according to the result of Cox analysis to enhance the prognostic ability of FNDC3B. Finally, the preliminary biological function of FNDC3B in PC cells was explored. RESULTS: The study demonstrated a significantly higher expression of FNDC3B in tumor tissues compared to normal pancreatic tissues, and this expression was significantly associated with various clinicopathological parameters. GSEA revealed the involvement of FNDC3B in biological processes and signaling pathways related to integrin signaling pathway and cell adhesion. Additionally, ssGSEA analysis indicated a positive correlation between FNDC3B expression and infiltration of Th2 cells and neutrophils, while showing a negative correlation with plasmacytoid dendritic cells and Th17 cells infiltration. Kaplan-Meier analysis further supported that high FNDC3B expression in PC patients was linked to shorter overall survival, disease-specific survival, and progression-free interval. However, although univariate analysis demonstrated a significant correlation between FNDC3B expression and prognosis in PC patients, this association did not hold true in multivariate analysis. Finally, our findings highlight the crucial role of FNDC3B expression in regulating proliferation, migration, and invasion abilities of PC cells. CONCLUSION: Despite limitations, the findings of this study underscored the potential of FNDC3B as a prognostic biomarker and its pivotal role in driving the progression of PC, particularly in orchestrating immune responses.

The discovery and evaluation of [18F]BMS-986229, a novel macrocyclic peptide PET radioligand for the measurement of PD-L1 expression and in-vivo PD-L1 target engagement.

PURPOSE: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. METHODS: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. RESULTS: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. CONCLUSION: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.

The role of miR-143-3p/FNDC1 axis on the progression of non-small cell lung cancer.

The study aimed to explore the functional role of fibronectin type III domain containing 1 (FNDC1) in nonsmall cell lung cancer (NSCLC), as well as the mechanism governing its expression. The expression levels of FNDC1 and related genes in tissue and cell samples were detected by qRT-PCR. Kaplan-Meier analysis was employed to analyze the association between FNDC1 level and the overall survival of NSCLC patients. Functional experiments such as CCK-8 proliferation, colony formation, EDU staining, migration and invasion assays were conducted to investigate the functional role of FNDC1 in regulating the malignancy of NSCLC cells. Bioinformatic tools and dual-luciferase reporter assay were used to identify the miRNA regulator of FNDC1 in NSCLC cells. Our data revealed the upregulation of FNDC1 at mRNA and protein levels in NSCLC tumor tissues cancer cell lines, compared with normal counterparts. NSCLC patients with higher FNDC1 expression suffered from a poorer overall survival. FNDC1 knockdown significantly suppressed the proliferation, migration and invasion of NSCLC cells, and had an inhibitory effect on tube formation. We further demonstrated that miR-143-3p was an upstream regulator of FNDC1 and miR-143-3p expression was repressed in NSCLC samples. Similar to FNDC1 knockdown, miR-143-3p overexpression inhibited the growth, migration and invasion of NSCLC cells. FNDC1 overexpression could partially rescue the effect of miR-143-3p overexpression.  FNDC1 silencing also suppressed the tumorigenesis of NSCLC cells in mouse model. In conclusion, FNDC1 promotes the malignant prototypes of NSCLC cells. miR-143-3p is a negative regulator of FNDC1 in NSCLC cells, which may serve as a promising therapeutic target in NSCLC.

Fibronectin type III domain-containing protein 5 promotes autophagy via the AMPK/mTOR signaling pathway in hepatocellular carcinoma cells, contributing to nab-paclitaxel chemoresistance.

Chemotherapy resistance is a huge challenge in the treatment of hepatocellular carcinoma because resistance to nab-paclitaxel largely affects the efficacy of chemotherapy. An increased expression of fibronectin type III domain-containing protein 5 (FNDC5) in hepatocellular carcinoma cells can predict post-hepatectomy complications in patients with hepatocellular carcinoma and also stimulate proliferation and invasion of hepatocellular carcinoma cells; however, its role in the chemotherapy of hepatocellular carcinoma cells has never been evaluated. Thus, this study aimed to explore whether FNDC5 regulates chemoresistance in hepatocellular carcinoma. We identified by immunohistochemistry that hepatocellular carcinoma tissues had a higher FNDC5 expression than normal tissues adjacent to the cancer cells. Subsequently, knockdown of FNDC5 in hepatocellular carcinoma cells resulted in their diminished resistance to cell death after chemotherapy with nab-paclitaxel. By contrast, overexpression of FNDC5 in hepatocellular carcinoma cells increased the resistance of hepatocellular carcinoma cells to treatment. Moreover, FNDC5 mechanistically promoted autophagy via the AMPK/mTOR signaling pathway, thereby reducing cell death induced by nab-paclitaxel. Finally, we tested our hypothesis by conducting animal experiments. In conclusion, FNDC5 could be used as a biomarker for predicting chemotherapeutic efficacy in hepatocellular carcinoma treated with nab-paclitaxel chemotherapy, and as a therapeutic target to overcome resistance to nab-paclitaxel in hepatocellular carcinoma chemotherapy.

Prognostic value of transcriptional expression of fibronectin type III domain-containing 4 (FNDC4) in head and neck carcinoma patients treated with chemoradiotherapy.

PURPOSE: FNDC4 gene encodes the fibronectin type III domain-containing 4 protein. Elevated expression of FNDC4 has been associated with poor prognosis in several types of cancer. There are no studies that have evaluated the prognostic capacity of FNDC4 in patients with head and neck cancer (HNSCC). The aim of our study was to analyze the relationship between the transcriptional expression of FNDC4 and prognosis in HNSCC patients. METHODS: We determined the transcriptional expression of FNDC4 in 67 patients with advanced-stage HNSCC (III-IV) treated with chemoradiotherapy. The FNDC4 expression was categorized according to the disease-specific survival with a recursive partitioning analysis. RESULTS: There were significant differences in disease-specific survival as a function of the level of FNDC4 transcriptional expression. The 5-year disease-specific survival for patients with high FNDC4 expression (n = 44, 65.7%) was 32.9% (95% CI: 16.5-49.3%), and for patients with low expression (n = 23, 34.3%) it was 85.4% (95% CI: 70.2-100%) (P = 0.0001). Patients with a high FNDC4 expression had poorer local (P = 0.097), regional (P = 0.008), and distant (0.034) recurrence-free survival. The results of a multivariate analysis showed that patients with a high FNDC4 expression had a 6.15-fold increased risk of death as a consequence of the HNSCC (95% CI: 1.71-22.06). CONCLUSION: FNCF4 transcriptional expression was significantly related to the disease-specific survival of HNSCC patients treated with chemoradiotherapy. Patients with elevated FNDC4 expression had a significant decrease in disease-specific survival.

Fibronectin type III domain-containing 5 improves aging-related cardiac dysfunction in mice.

Aging is an important risk factor for cardiovascular diseases, and aging-related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin-induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno-associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging-related cardiac dysfunction. To verify the involvement of nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) and AMP-activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9-FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor-α (TNF-α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac-specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging-related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF-α-stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon-like peptide-1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging-related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1ß, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.

Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis.

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-MertkFnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

Avidity and Cell Uptake of Integrin-Targeting Polypeptide Micelles is Strongly Shape-Dependent.

We describe a genetically encoded micelle for targeted delivery consisting of a diblock polypeptide with segments derived from repetitive protein motifs inspired by Drosophila melanogaster Rec-1 resilin and human tropoelastin with a C-terminal fusion of an integrin-targeting fibronectin type III domain. By systematically varying the weight fraction of the hydrophilic elastin-like polypeptide (ELP) block and molecular weight of the diblock polypeptide, we designed micelles of different morphologies that modulate the binding avidity of the human wild-type 10th fibronectin domain (Fn3) as a function of shape. We show that wormlike micelles that present the Fn3 domain have a 1000-fold greater avidity for the αvß3 receptor compared to the monomer ligand and an avidity that is greater than a clinically relevant antibody that is driven by their multivalency. The amplified avidity of these micelles leads to significantly increased cellular internalization, a feature that may have utility for the intracellular delivery of drugs that are loaded into the core of these micelles.

Design of fibronectin type III domains fused to an elastin-like polypeptide for the osteogenic differentiation of human mesenchymal stem cells.

Extracellular matrix (ECM) including fibronectin (FN) and elastin plays a pivotal role in providing a microenvironment to support tissue regeneration in stem cell therapy. To develop a novel biomimetic ECM for stem cell differentiation, we engineered FN type III 9 and 10 domains fused to elastin-like polypeptides (FN-ELPs). The recombinant FN-ELP fusion protein was expressed in Escherichia coli and purified by inverse transition cycling. Human mesenchymal stem cells (hMSCs) cultured on plates coated with FN-ELP had significantly greater adhesion activity and proliferation than cells grown on non-coated plates. FN-ELP induced the osteogenic differentiation by elevating alkaline phosphatase (ALP) and mineralization activity of hMSCs. Furthermore, the osteogenic marker gene expressions of ALP, collagen type I (Col I), osteopontin (OPN), and transcriptional coactivator with a PDZ-binding motif (TAZ) were increased in hMSCs cultured on plates coated with FN-ELP. We reported a novel biomimetic ECM with potential for bone regeneration that promotes the osteogenic differentiation of hMSCs.

FN3 Protein Conjugates for Cancer Diagnosis and Imaging Studies.

Bioconjugation of biologically useful proteins is in great demand (e.g., conjugation to biotins, metal chelators, and drug carriers to target specific tissues for both in vitro and in vivo use). These conjugates provide widespread opportunities for various biological and biomedical applications. Evolving state-of-the-art protein conjugation strategies have led to the development of many affinity ligands, including for cancer imaging and diagnosis. However, to achieve the desirable protein conjugates, there are many challenges that remain to be addressed in order to obtain a reproducible procedure for all proteins and ligands. These include a control over the protein modification and the efficiency of the conjugation while retaining the original biological protein affinity postmodification. Here we present detailed conjugation methods for the human fibronectin tenth type III domain (FN3) protein scaffold for use in preclinical PET imaging. More specifically, this chapter provides detailed methods to produce a FN3 and a FN3-chelator-conjugate, its labeling with the radionuclide 64-Cu, and its use for noninvasive PET imaging in mice.

Allosteric modulation of a human protein kinase with monobodies.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Site-specific HNK-1 epitope on alternatively spliced fibronectin type-III repeats in tenascin-C promotes neurite outgrowth of hippocampal neurons through contactin-1.

The human natural killer-1 (HNK-1) carbohydrate epitope, composed of a unique sulfated trisaccharide (HSO3-3GlcAß1-3Galß1-4GlcNAc-R), is highly expressed during brain development and regulates higher brain function. However, it remains unclear which glycoprotein carries the HNK-1 epitope in the embryonic brain and the functional role it plays. Here, we showed that one of the major HNK-1 carrier proteins in the embryonic brain is tenascin-C (TNC), an extracellular matrix protein that regulates neurite outgrowth by interacting with the GPI-anchored protein contactin-1 (CNTN). Because the alternatively spliced fibronectin type-III (FNIII) repeats in TNC give rise to many isoforms and affect neuronal function, we evaluated neurite outgrowth of primary hippocampal neurons on purified recombinant FNIII repeats with or without the HNK-1 epitope as a substrate. We found that the presence of the HNK-1 epitope on the C domain of TNC promoted neurite outgrowth, and that this signal was mediated by CNTN, which is an HNK-1-expressing neuronal receptor. The neurite-promoting activity of the HNK-1 epitope on TNC required neuronal HNK-1 expression, which was defective in neurons lacking the glucuronyltransferases GlcAT-P and GlcAT-S. These results suggest that the HNK-1 epitope is a key modifier of TNC and CNTN in the regulation of embryonic brain development.

A Novel Engineered Small Protein for Positron Emission Tomography Imaging of Human Programmed Death Ligand-1: Validation in Mouse Models and Human Cancer Tissues.

PURPOSE: To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens.Experimental Design: The hPD-L1 protein binder, FN3hPD-L1, was engineered using a 12-kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64Cu-FN3hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice (n = 3-5/group) were imaged with 64Cu-FN3hPD-L1 PET imaging (1-24 hours postinjection of 3.7 MBq/7 µg of Do-FN3 in 200 µL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (ii) CT26/hPD-L1 tumors blocked (blk) by preinjected nonradioactive FN3hPD-L1 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) MDA-MB-231 xenografts. The FN3hPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens. RESULTS: FN3hPD-L1 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. 64Cu-FN3hPD-L1 radiotracer showed >70% yield and >95% purity. 64Cu-FN3hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6 ± 0.9 and 13.1 ± 2.3 at 1 and 4 hours postinjection, respectively. The FN3hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies. CONCLUSIONS: The 64Cu-FN3hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

The Structure in Solution of Fibronectin Type III Domain 14 Reveals Its Synergistic Heparin Binding Site.

Fibronectin is a large multidomain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells [Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818; McCarthy, J. B., et al. (1988) Biochemistry 27, 1380-1388; Drake, S. L., et al. (1993) J. Biol. Chem. 268, 15859-15867]. The stronger heparin/HS binding site on fibronectin, Hep-II, spans fibronectin type III domains 12-14. Previous site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation, and crystallographic structural studies all agree that the main heparin binding site is located on the surface of fibronectin type III domain 13 [Ingham, K. C., et al. (1993) Biochemistry 32, 12548-12553; Sharma, A., et al. (1999) EMBO J. 18, 1468-1479; Sachchidanand, L. O., et al. (2002) J. Biol. Chem. 277, 50629-50635]. However, the "synergy site" for heparin binding located on fibronectin type III domain 14 remained elusive because the actual binding sites could not be identified. Using NMR spectroscopy and isothermal titration calorimetry, we show here that heparin is able to bind to a cationic 'cradle' of fibronectin type III domain 14 formed by the PRARI sequence, which is involved in the integrin α4ß1 interaction [Mould, A. P., and Humphries, M. J. (1991) EMBO J. 10, 4089-4095], and to the flexible loop comprising residues KNNQKSE between the last two ß-strands, D and E, of FN14. Our data reveal that the individual FN14 domain binds to the sulfated sugars Dp8 and Reviparin with affinities similar to those of the individual domain FN13 [Breddin, H. K. (2002) Expert Opin. Pharmacother. 3, 173-182]. It is noteworthy that by introduction of the last ß-strand of FN13 and the linker region between FN type III domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 involves only the KNNQKSE sequence.

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.

Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding.

Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics.

Amelioration of collagen antibody induced arthritis in mice by an antibody directed against the fibronectin type III repeats of tenascin-C: Targeting fibronectin type III repeats of tenascin-C in rheumatoid arthritis.

Tenascin-C (TN-C) levels are elevated in the synovial tissue and fluid, as well as cartilage of rheumatoid arthritis (RA) patients. In addition, the presence of TN-C fragments has also been documented in arthritic cartilage. We have previously shown that a single chain variable fragment antibody (TN64), directed against the fibronectin type III repeats 1-5 (TNfnIII 1-5) of TN-C, effectively inhibits fibrotic pathology. Given that fibrosis results from chronic inflammation, and the fact that increased levels of TN-C in the synovial fluid of patients with RA contributes to synovial inflammation and joint destruction, we aimed to investigate the role of TNfnIII 1-5 region of TN-C in RA pathogenesis. Using either the wild type or variants of the two integrin-binding motifs (RGD and AEIDGIEL) present within the TNfnIII 1-5 polypeptide, we demonstrate that the adhesion and migration of synovial fibroblasts is RGD-dependent. The antibody TN64 is effective in inhibiting migration of cells in response to TnfnIII 1-5, and prevents fibroblast-mediated destruction of cartilage. The TN64 antibody was further tested in collagen antibody induced arthritic (CAIA) mice. Our data shows the efficacy of TN64 in preventing induction of arthritis, with significant downregulation of RA-associated cytokines. This suggests that components of the extracellular matrix such as the TNfnIII 1-5 region of TN-C could be exploited to develop therapies to suppress inflammation seen in RA. The TN64 antibody is one such promising candidate in the development of novel treatments for RA.

DCC/NTN1 complex mutations in patients with congenital hypogonadotropic hypogonadism impair GnRH neuron development.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice.