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

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

Engineered Fn3 Protein has Targeted Therapeutic Effect on Mesothelin-Expressing Cancer Cells and Increases Tumor Cell Sensitivity to Chemotherapy

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin

Protein-Polymer Conjugates Synthesized Using Water-Soluble Azlactone-Functionalized Polymers Enable Receptor-Specific Cellular Uptake Toward Targeted Drug Delivery

Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein–polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small-molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone), synthesized using reversible addition–fragmentation chain transfer polymerization, was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein–polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin–polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein–polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein–polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications

Targeting a highly repetitive genomic sequence for sensitive and specific molecular detection of the filarial parasite Mansonella perstans from human blood and mosquitoes

Background: Mansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions. Methodology/Principal findings: Utilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as “negative” using the less sensitive detection method. Conclusions/Significance: Through the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance

Integrated xenosurveillance of Loa loa, Wuchereria bancrofti, Mansonella perstans and Plasmodium falciparum using mosquito carcasses and faeces: A pilot study in Cameroon.

Background Community presence of loiasis must be determined before mass drug administration programmes for lymphatic filariasis and onchocerciasis can be implemented. However, taking human blood samples for loiasis surveillance is invasive and operationally challenging. A xenosurveillance approach based on the molecular screening of mosquitoes and their excreta/feces (E/F) for Loa loa DNA may provide a non-invasive method for detecting the community presence of loiasis. Methods We collected 770 wild mosquitoes during a pilot study in a known loiasis transmission area in Mbalmayo, Cameroon. Of these, 376 were preserved immediately while 394 were kept in pools to collect 36-hour E/F samples before processing. Carcasses and E/F were screened for L. loa DNA. To demonstrate this method’s potential for integrated disease surveillance, the samples were further tested for Wuchereria bancrofti, Mansonella perstans, and Plasmodium falciparum. Results Despite limited sample numbers, L. loa DNA was detected in eight immediately-stored mosquitoes (2.13%; 95% CI 1.08 to 4.14), one carcass stored after providing E/F (0.25%; 95% CI 0.04 to 1.42), and three E/F samples (estimated prevalence 0.77%; 95% CI 0.15 to 2.23%). M. perstans and P. falciparum DNA were also detected in carcasses and E/F samples, while W. bancrofti DNA was detected in E/F. None of the carcasses positive for filarial worm DNA came from pools that provided a positive E/F sample, supporting the theory that, in incompetent vectors, ingested parasites undergo a rapid, complete expulsion in E/F. Conclusions Mosquito xenosurveillance may provide a useful tool for the surveillance of loiasis alongside other parasitic diseases

Measurement of the t t-bar production cross section in the dilepton channel in pp collisions at sqrt(s) = 7 TeV

The t t-bar production cross section (sigma[t t-bar]) is measured in proton-proton collisions at sqrt(s) = 7 TeV in data collected by the CMS experiment, corresponding to an integrated luminosity of 2.3 inverse femtobarns. The measurement is performed in events with two leptons (electrons or muons) in the final state, at least two jets identified as jets originating from b quarks, and the presence of an imbalance in transverse momentum. The measured value of sigma[t t-bar] for a top-quark mass of 172.5 GeV is 161.9 +/- 2.5 (stat.) +5.1/-5.0 (syst.) +/- 3.6(lumi.) pb, consistent with the prediction of the standard model.Comment: Replaced with published version. Included journal reference and DO

Search for anomalous t t-bar production in the highly-boosted all-hadronic final state

A search is presented for a massive particle, generically referred to as a Z', decaying into a t t-bar pair. The search focuses on Z' resonances that are sufficiently massive to produce highly Lorentz-boosted top quarks, which yield collimated decay products that are partially or fully merged into single jets. The analysis uses new methods to analyze jet substructure, providing suppression of the non-top multijet backgrounds. The analysis is based on a data sample of proton-proton collisions at a center-of-mass energy of 7 TeV, corresponding to an integrated luminosity of 5 inverse femtobarns. Upper limits in the range of 1 pb are set on the product of the production cross section and branching fraction for a topcolor Z' modeled for several widths, as well as for a Randall--Sundrum Kaluza--Klein gluon. In addition, the results constrain any enhancement in t t-bar production beyond expectations of the standard model for t t-bar invariant masses larger than 1 TeV.Comment: Submitted to the Journal of High Energy Physics; this version includes a minor typo correction that will be submitted as an erratu