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Sloth, Ane Beth, Babak Bakhshinejad, Malte Jensen, Camilla Stavnsbjerg, Mikkel Baldtzer Liisberg, Maria Rossing, and Andreas Kjaer. "Analysis of Compositional Bias in a Commercial Phage Display Peptide Library by Next-Generation Sequencing." Viruses 14, no. 11 (October 29, 2022): 2402. http://dx.doi.org/10.3390/v14112402.
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The principal presumption of phage display biopanning is that the naïve library contains an unbiased repertoire of peptides, and thus, the enriched variants derive from the affinity selection of an entirely random peptide pool. In the current study, we utilized deep sequencing to characterize the widely used Ph.DTM-12 phage display peptide library (New England Biolabs). The next-generation sequencing (NGS) data indicated the presence of stop codons and a high abundance of wild-type clones in the naïve library, which collectively result in a reduced effective size of the library. The analysis of the DNA sequence logo and global and position-specific frequency of amino acids demonstrated significant bias in the nucleotide and amino acid composition of the library inserts. Principal component analysis (PCA) uncovered the existence of four distinct clusters in the naïve library and the investigation of peptide frequency distribution revealed a broad range of unequal abundances for peptides. Taken together, our data provide strong evidence for the notion that the naïve library represents substantial departures from randomness at the nucleotide, amino acid, and peptide levels, though not undergoing any selective pressure for target binding. This non-uniform sequence representation arises from both the M13 phage biology and technical errors of the library construction. Our findings highlight the paramount importance of the qualitative assessment of the naïve phage display libraries prior to biopanning.
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Sabir, Jamal S. M., Ahmed Atef, Fotouh M. El-Domyati, Sherif Edris, Nahid Hajrah, Ahmed M. Alzohairy, and Ahmed Bahieldin. "Construction of naïve camelids VHH repertoire in phage display-based library." Comptes Rendus Biologies 337, no. 4 (April 2014): 244–49. http://dx.doi.org/10.1016/j.crvi.2014.02.004.
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Sommavilla, R., V. Lovato, A. Villa, D. Sgier, and D. Neri. "Design and construction of a naïve mouse antibody phage display library." Journal of Immunological Methods 353, no. 1-2 (February 2010): 31–43. http://dx.doi.org/10.1016/j.jim.2010.01.003.
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Wagner, Hanna, Sarah Wehrle, Etienne Weiss, Marco Cavallari, and Wilfried Weber. "A Two-Step Approach for the Design and Generation of Nanobodies." International Journal of Molecular Sciences 19, no. 11 (November 2, 2018): 3444. http://dx.doi.org/10.3390/ijms19113444.
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Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.
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Kamstrup Sell, Danna, Ane Beth Sloth, Babak Bakhshinejad, and Andreas Kjaer. "A White Plaque, Associated with Genomic Deletion, Derived from M13KE-Based Peptide Library Is Enriched in a Target-Unrelated Manner during Phage Display Biopanning Due to Propagation Advantage." International Journal of Molecular Sciences 23, no. 6 (March 18, 2022): 3308. http://dx.doi.org/10.3390/ijms23063308.
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The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.TM-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count- and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage.
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Killeen, G. F., B. D. Foy, R. H. Frohn, D. Impoinvil, A. Williams, and J. C. Beier. "Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning withAnopheles gambiae(Diptera: Culicidae) midgut homogenate." Bulletin of Entomological Research 93, no. 1 (January 2003): 31–37. http://dx.doi.org/10.1079/ber2002216.
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AbstractA high diversity library of recombinant human antibodies was selected on complex antigen mixtures from midguts of femaleAnopheles gambiaeGiles. The library of phage-displayed single chain variable region fragment constructs, derived from β-lymphocyte mRNA of naïve human donors, was repeatedly selected and reamplified on the insoluble fraction of midgut homogenates. Five rounds of panning yielded only one midgut-specific clone, which predominated the resulting antibody panel. InA. gambiae, the epitope was found throughout the tissues of females but was absent from the midgut of males. The cognate antigen proved to be detergent soluble but very sensitive to denaturation and could not be isolated or identified by Western blot of native electrophoresis gels or by immunoprecipitation. Nevertheless, immunohistology revealed that this sex-specific epitope is associated with the lumenal side of the midgut. Severe bottlenecking may limit the utility of phage display selection from naïve libraries for generating diverse panels of antibodies against complex mixtures of antigens from insect tissues. These results suggest that the selection of sufficiently diverse antibody panels, from which mosquitocidal or malaria transmission-blocking antibodies can be isolated, may require improved selection methods or specifically enriched pre-immunized libraries.
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Qiu, Yu-Lou, Qing-Hua He, Yang Xu, Arun K. Bhunia, Zhui Tu, Bo Chen, and Yuan-Yuan Liu. "Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay." Analytica Chimica Acta 887 (August 2015): 201–8. http://dx.doi.org/10.1016/j.aca.2015.06.033.
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Park, Sae-Gwang, Yong-Joo Jeong, Yong-Yi Lee, Ik-Jung Kim, Su-Kil Seo, Eui-Joong Kim, Heung-Chae Jung, et al. "Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naïve antibody phage library." Antiviral Research 68, no. 3 (December 2005): 109–15. http://dx.doi.org/10.1016/j.antiviral.2005.06.012.
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English, Hejiao, Jessica Hong, and Mitchell Ho. "Ancient species offers contemporary therapeutics: an update on shark VNAR single domain antibody sequences, phage libraries and potential clinical applications." Antibody Therapeutics 3, no. 1 (January 2020): 1–9. http://dx.doi.org/10.1093/abt/tbaa001.
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ABSTRACT The antigen binding variable domain (VNAR) of the shark immunoglobulin new antigen receptor (IgNAR) evolved approximately 500 million years ago and it is one of the smallest antibody fragments in the animal kingdom with sizes of 12–15 kDa. This review discusses the current knowledge of the shark VNAR single domain sequences and ongoing development of shark VNARs as research tools as well as potential therapeutics, in particular highlighting the recent next-generation sequencing analysis of 1.2 million shark VNAR sequences and construction of a large phage displayed shark VNAR library from six naïve adult nurse sharks (Ginglymostoma cirratum). The large phage-displayed VNAR single domain library covers all the four known VNAR types (Types I–IV) and many previously unknown types. Ongoing preclinical development will help define the utility of shark VNAR single domains as a potentially new family of drug candidates for treating cancer and other human diseases.
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Feng, Mingqian, Hejiao Bian, Xiaolin Wu, Tianyun Fu, Ying Fu, Jessica Hong, Bryan D. Fleming, Martin F. Flajnik, and Mitchell Ho. "Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naïve nurse sharks." Antibody Therapeutics 2, no. 1 (November 7, 2018): 1–11. http://dx.doi.org/10.1093/abt/tby011.
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ABSTRACT Background Shark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. Methods Here, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named “EASeL”) to construct a large VNAR antibody library with a size of 1.2 × 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum). Results The next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I–IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (PE38-B6) has a high affinity (Kd = 10.1 nM) for its antigen. Conclusions The naïve nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries.
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Naranjo, Leslie, Fortunato Ferrara, Nicolas Blanchard, Caroline Demangel, Sara D’Angelo, M. Frank Erasmus, Andre A. Teixera, and Andrew R. M. Bradbury. "Recombinant Antibodies against Mycolactone." Toxins 11, no. 6 (June 17, 2019): 346. http://dx.doi.org/10.3390/toxins11060346.
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In the past, it has proved challenging to generate antibodies against mycolactone, the primary lipidic toxin A of Mycobacterium ulcerans causing Buruli ulcer, due to its immunosuppressive properties. Here we show that in vitro display, comprising both phage and yeast display, can be used to select antibodies recognizing mycolactone from a large human naïve phage antibody library. Ten different antibodies were isolated, and hundreds more identified by next generation sequencing. These results indicate the value of in vitro display methods to generate antibodies against difficult antigenic targets such as toxins, which cannot be used for immunization unless inactivated by structural modification. The possibility to easily generate anti-mycolactone antibodies is an exciting prospect for the development of rapid and simple diagnostic/detection methods.
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Liu, Chang C., Antha V. Mack, Meng-Lin Tsao, Jeremy H. Mills, Hyun Soo Lee, Hyeryun Choe, Michael Farzan, Peter G. Schultz, and Vaughn V. Smider. "Protein evolution with an expanded genetic code." Proceedings of the National Academy of Sciences 105, no. 46 (November 11, 2008): 17688–93. http://dx.doi.org/10.1073/pnas.0809543105.
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We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.
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Parray, Hilal Ahmad, Adarsh Kumar Chiranjivi, Shailendra Asthana, Naveen Yadav, Tripti Shrivastava, Shailendra Mani, Chandresh Sharma, et al. "Identification of an anti–SARS–CoV-2 receptor-binding domain–directed human monoclonal antibody from a naïve semisynthetic library." Journal of Biological Chemistry 295, no. 36 (July 29, 2020): 12814–21. http://dx.doi.org/10.1074/jbc.ac120.014918.
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There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS– coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)–interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.
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Hjelm, Linnea Charlotta, Hanna Lindberg, Stefan Ståhl, and John Löfblom. "Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides." International Journal of Molecular Sciences 24, no. 1 (January 3, 2023): 836. http://dx.doi.org/10.3390/ijms24010836.
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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.
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McElhiney, J. "Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library." FEMS Microbiology Letters 193, no. 1 (December 1, 2000): 83–88. http://dx.doi.org/10.1016/s0378-1097(00)00460-2.
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Norbury, Luke J., Katarzyna Basałaj, Piotr Bąska, Anna Zawistowska-Deniziak, Alicja Kalinowska, Przemysław Wilkowski, Agnieszka Wesołowska, and Halina Wędrychowicz. "Generation of a single-chain variable fragment phage display antibody library from naïve mice panned against Fasciola hepatica antigens." Experimental Parasitology 205 (October 2019): 107737. http://dx.doi.org/10.1016/j.exppara.2019.107737.
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Alfaleh, Mohamed, Neetika Arora, Michael Yeh, Christopher de Bakker, Christopher Howard, Philip Macpherson, Rachel Allavena, et al. "Canine CD117-Specific Antibodies with Diverse Binding Properties Isolated from a Phage Display Library Using Cell-Based Biopanning." Antibodies 8, no. 1 (February 12, 2019): 15. http://dx.doi.org/10.3390/antib8010015.
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CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.
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Chisholm, Maia, Laura Quigley, James McMahon, Toshiyuki Mori, Barbara Giomarelli, Valance Washington, and Daniel McVicar. "Inhibition of thrombin-induced platelet aggregation using human single-chain Fv antibodies specific for TREM-like transcript-1." Thrombosis and Haemostasis 97, no. 06 (2007): 955–63. http://dx.doi.org/10.1160/th06-08-0456.
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SummaryTREM-like transcript-1 (TLT-1) is a novel platelet membrane receptor, which has been recently characterized in mice. TLT-1 is expressed exclusively in platelets and megakaryocytes, and its expression is dramatically upregulated upon platelet activation, suggesting that it plays a unique role in hemostasis and/or thrombosis. In this study we identified and characterized highly specific human monoclonal antibodies that bind to TLT-1 by screening a naïve library of single chain Fv fragments (scFvs) displayed on filamentous phage (Thomlinson I library). These scFvs detected plate-bound TLT-1, captured soluble TLT-1, and readily reacted with cell-bound TLT-1 on transfectants and primary human platelets. Most importantly, anti-TLT-1 scFvs inhibited thrombin-mediated human platelet aggregation. This inhibition was specific for thrombin-induced aggregation and was reversible with higher doses of agonist. These data are the first to demonstrate a biological role for TLT-1 and its potential as a therapeutic target. The human scFvs isolated in this study may represent novel anti-platelet therapeutic agents.
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Stewart, Christine S., C. Roger MacKenzie, and J. Christopher Hall. "Isolation, characterization and pentamerization of α-cobrotoxin specific single-domain antibodies from a naïve phage display library: Preliminary findings for antivenom development." Toxicon 49, no. 5 (April 2007): 699–709. http://dx.doi.org/10.1016/j.toxicon.2006.11.023.
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Guzmán-Bringas, Omar U., Keyla M. Gómez-Castellano, Edith González-González, Juana Salinas-Trujano, Said Vázquez-Leyva, Luis Vallejo-Castillo, Sonia M. Pérez-Tapia, and Juan C. Almagro. "Discovery and Optimization of Neutralizing SARS-CoV-2 Antibodies Using ALTHEA Gold Plus Libraries™." International Journal of Molecular Sciences 24, no. 5 (February 27, 2023): 4609. http://dx.doi.org/10.3390/ijms24054609.
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We recently reported the isolation and characterization of anti-SARS-CoV-2 antibodies from a phage display library built with the VH repertoire of a convalescent COVID-19 patient, paired with four naïve synthetic VL libraries. One of the antibodies, called IgG-A7, neutralized the Wuhan, Delta (B.1.617.2) and Omicron (B.1.1.529) strains in authentic neutralization tests (PRNT). It also protected 100% transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from SARS-CoV-2 infection. In this study, the four synthetic VL libraries were combined with the semi-synthetic VH repertoire of ALTHEA Gold Libraries™ to generate a set of fully naïve, general-purpose, libraries called ALTHEA Gold Plus Libraries™. Three out of 24 specific clones for the RBD isolated from the libraries, with affinity in the low nanomolar range and sub-optimal in vitro neutralization in PRNT, were affinity optimized via a method called “Rapid Affinity Maturation” (RAM). The final molecules reached sub-nanomolar neutralization potency, slightly superior to IgG-A7, while the developability profile over the parental molecules was improved. These results demonstrate that general-purpose libraries are a valuable source of potent neutralizing antibodies. Importantly, since general-purpose libraries are “ready-to-use”, it could expedite isolation of antibodies for rapidly evolving viruses such as SARS-CoV-2.
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Aliprandi, Marisa, Eleonora Sparacio, Flavia Pivetta, Giuseppe Ossolengo, Roberta Maestro, and Ario de Marco. "The Availability of a Recombinant Anti-SNAP Antibody in VHH Format Amplifies the Application Flexibility of SNAP-Tagged Proteins." Journal of Biomedicine and Biotechnology 2010 (2010): 1–7. http://dx.doi.org/10.1155/2010/658954.
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Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.
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Pedchenko, T. V., R. Mernaugh, and P. P. Massion. "Tumor specific antibodies for the early diagnosis of lung cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 18068. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.18068.
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18068 Background: The survival rates in lung cancer vary significantly by stage, the earlier the better. However, only 16% of patients are diagnosed at stage I. This implies an urgent need to identify a panel of biomarkers for the early detection of lung cancer in order to offer an improve chance for cure. We hypothesized that a unique phage-display recombinant antibody library approach will allow us to capture from serum and evaluate cancer antibodies as novel diagnostic biomarkers of non-small cell lung cancer. Methods: Immunoglobulins G (IgG) were purified from 3 groups of pooled serum: 10 diagnosed with adenocarcinoma, 10 squamous carcinomas, and 10 controls. A naïve rodent scFv (single chain fragment variable) phage-displayed recombinant antibody library (∼2.9 x 109 members) was used to select scFv specific for cancer human IgG. Individual bacterial colonies obtained after two rounds of subtractive biopanning on purified normal IgG and cancer IgG were picked and induced to express soluble E-tagged ScFv antibodies. An ELISA was used to screen ScFv for binding activity to pooled normal and cancer serum IgG. Single scFv clones that showed preferential binding to cancer IgG in ELISA were further validated for binding specificity by dot blot. ScFv were assayed against individual serum samples obtained from 120 normal, 60 adenocarcinoma, 56 squamous carcinoma, and 35 other, non-small cell, lung cancers. Dot blot signal intensity was quantified using Typhoon 8600 imaging system. Results: Seventy-five individual bacterial clones produced scFv that, by ELISA, distinguished purified normal from cancer IgG from pooled serum samples. Selected scFv antibodies show significantly higher binding activity to cancer-related IgG as compared to normal IgG. Twenty-five scFv were further validated for binding activity in individual human serum samples by dot blot assays. Ten scFv clearly differentiated cancer immunoglobulins from normal in human serum by dot blot, with preferential binding either to adeno- or squamous carcinoma. Conclusions: In this preliminary dataset, we identified scFv antibodies that interact with immunoglobulins present in human cancer serum samples. We show that this is a feasible and promising approach to discover cancer-specific immunoglobulins as candidate diagnostic biomarkers. No significant financial relationships to disclose.
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Brichta, J., H. Vesela, and M. Franek. "Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library." Veterinární Medicína 48, No. 9 (March 30, 2012): 237–47. http://dx.doi.org/10.17221/5776-vetmed.
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Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.
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Zhang, Xiao, Chongxin Xu, Cunzheng Zhang, Yuan Liu, Yajing Xie, and Xianjin Liu. "Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library." Toxicon 81 (April 2014): 13–22. http://dx.doi.org/10.1016/j.toxicon.2014.01.010.
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Tan, Tyng Hwey, Elizabeth Patton, Carol A. Munro, Dora E. Corzo-Leon, Andrew J. Porter, and Soumya Palliyil. "Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein." Viruses 13, no. 11 (November 2, 2021): 2201. http://dx.doi.org/10.3390/v13112201.
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ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.
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Yoon, Jeong Heon, Anja Schmidt, Yong Chan Kim, Christoph Koenigs, and David William Scott. "Immunosuppressive FVIII-Specific Human Cartregs in Hemophilia a." Blood 126, no. 23 (December 3, 2015): 291. http://dx.doi.org/10.1182/blood.v126.23.291.291.
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Abstract Hemophilia A is an X-linked disorder, in which mutations in the coagulation Factor VIII (FVIII) gene lead to a loss of FVIII function and serious bleeding episodes. These episodes can be treated with recombinant FVIII protein replacement. Unfortunately, ~25% of hemophilia A patients produce inhibitory anti-FVIII antibodies because of lack of tolerance. Thus, it is necessary to develop effective tolerogenic therapies to prevent, as well as reverse, inhibitor formation. Previously, we generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T cell clone. The resulting engineered T cells bind MHC tetramers, proliferate in response to a specific FVIII epitope, and suppress effector responses to FVIII. In this study, we engineered a FVIII-specific chimeric antigen receptor (ANS8CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Following initial experiments in naïve CD4 T cells, this CAR was introduced into human Tregs. Western blot and specific staining with FVIII verified CAR expression. Transduced ANS8CAR Tregs proliferated in response to FVIII and were able to suppress the proliferation of FVIII-specific T effector cells in vitro. Additionally, the proliferation of T effector cells with different FVIII domain specificity was suppressed as well when ANS8CAR-transduced Tregs were activated with FVIII. Thus, engineered cells are able to promote bystander suppression. Cytokine expression of ANS8CAR-transduced Tregs was comparable to expression of untransduced and TCR-transduced Tregs indicating that the regulatory phenotype of Tregs was not negatively influenced by ANS8CAR expression. In conclusion, CAR-transduced Tregs seem to be a promising alternative to TCR-transduced Tregs for a future tolerogenic treatment of hemophilia A patients with inhibitory FVIII-specific antibodies. Supported by NIH grants HL061883 and HL126727 (DWS), the Society of Thrombosis and Hemostasis Research, and the Günter Landbeck Excellence Award (AS). Disclosures Kim: Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
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Naumann, Anja, Yongchan Kim, Christoph Königs, and David W. Scott. "Generation and Characterization of FVIII-Specific CAR-Transduced Regulatory T Cells." Blood 124, no. 21 (December 6, 2014): 236. http://dx.doi.org/10.1182/blood.v124.21.236.236.
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Abstract The development of inhibitory antibodies (inhibitors) against FVIII is the most critical complication in the treatment of hemophilia A patients as hemostasis can no longer be reestablished by FVIII replacement therapy. Immune tolerance induction by frequent FVIII infusions is demanding, costly and not successful in all treated patients leading to an urgent need for the development of new therapeutic approaches for the prevention or treatment of FVIII inhibitors. Regulatory T cells (Tregs) are important for the maintenance of tolerance and have a high therapeutic potential in the context of autoimmune or inflammatory immune disorders. As Tregs are polyclonal, treatment with Treg pools comprises the risk of a general immunosuppression. Thus, the establishment of antigen-specific Tregs could be of great benefit for a broad range of patients, including inhibitor positive hemophilia A patients. To create such specific Tregs, a FVIII-specific scFv isolated out of a synthetic phage display library was used to generate a second generation chimeric antigen receptor (CAR). To verify the specificity of the CAR for FVIII and the functionality of the recombined cytoplasmic domain (CD28 and CD3zeta), naïve CD4 T cells were retrovirally transduced with the generated CAR construct and a proliferation assay was conducted in the presence of plate-bound or soluble FVIII, as well as soluble FVIII presented by autologous irradiated PBMCs. Proliferation of transduced cells was more effective when FVIII was presented plate-bound or by PBMCs. In a therapeutically-relevant setting, this would be very promising, as transduced T cells should not be activated by soluble FVIII in the bloodstream but rather by FVIII presented on antigen-presenting cells in lymphatic organs. Next, functionality of the CAR construct in Tregs was addressed. Transduced Tregs showed extracellular expression of the scFv and could be stimulated MHC-independently with FVIII. Such stimulated cells showed increased expression of Treg activation markers LAP and GARP. Thus, by using the generated CAR for transduction of Tregs, it was possible to create FVIII-specific Tregs that can be stimulated MHC-independently, opening new possibilities for therapeutic approaches in hemophilia A patients with FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.
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Kolluri, Aarti, Dan Li, Nan Li, and Mitchell Ho. "Engineered, fully human nanobody-based CAR T cells have enhanced antitumor activity against hepatocellular carcinoma in preclinical models." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e14512-e14512. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e14512.
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e14512 Background: Glypican 3 (GPC3), a cell surface protein associated with early development, is an established biomarker of hepatocellular carcinoma (HCC). In a vast compendium of work, HN3, a human GPC3 nanobody (VH) isolated from a phage library, was shown to abrogate HCC tumors and block the distally located Wnt binding domain of GPC3. To date, it remains challenging how to precisely engineer CAR T cells for improved efficacy in solid tumors. In the context of hepatocellular carcinoma, we determined how hinges and transmembrane portions of varying structures and sizes affect CAR T cell function. Here, we show how rationally designed, engineered Chimeric Antigen Receptor (CAR) T cells containing HN3, demonstrate enhanced CAR T activity in HCC. Methods: We generated and compared multiple permutations of GPC3 targeted CAR T cells containing HN3, CD8, CD28, IgG4 and Fc domains. Incorporation of HN3, into multiple CAR formats yielded nine constructs, which were screened for antigen-specific and antigen-independent signaling using a luciferase reporter system in Hep3B, Huh7 and HepG2 cell lines. For in vivo assessment, immunodeficient NSG mice were intraperitonially injected with Hep3B or Huh7 cells and treated with a single dose (5 or 15 million) of engineered HN3 CAR T cells. Blood was collected at 2, 4 and 5 weeks to assess proliferation, T cell subtypes and exhaustion. Results: In vitro, the HN3-IgG4H-CD28TM CAR T cells induced high specificity and cytotoxic activity. HN3-IgG4H-CD28TM CAR T cells markedly improved HN3 cell killing activity by 30-40% in low (1.6:1) and high (25:1) effector to target ratios. Similarly, in vivo, in a high antigen environment, two determinative changes in the hinge region and transmembrane domains led to complete tumor eradication in immunodeficient mice bearing HCC tumors within 7-10 days. Moreover, HN3-IgG4H-CD28TM CAR T cells maintained specific tumor killing and averted exhaustion by producing a clear T cell response signature of enriched cytotoxic-memory (Temra) CD8+ along with a subset of naïve T cells. Conclusions: Engineered nanobody based CAR T cells containing the appropriate hinge and transmembrane domains can lead to determinative T cell signaling capable of inducing swift and durable eradication of HCC tumors. Altogether, we show that engineered HN3 CAR T cell therapy demonstrates strong potential for treatment of aggressive HCCs enabled by Wnt dysregulation.
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Lee, Kyung-Sik, Hye-Seong Park, Guk-Yeol Park, Gi-Chan Lee, Eun-Ji Choi, Jae-in Yu, Seung-Joo Yang, et al. "Abstract 6189: Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6189. http://dx.doi.org/10.1158/1538-7445.am2022-6189.
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Abstract [Purpose] Immunosuppressive tumor microenvironment (TME) is the major hurdle to cancer immunotherapy. Advanced hepatocellular carcinoma (aHCC) is one of the representative cold tumors with immunosuppressive TME and presents obvious correlation between immune check point expression, such as programmed cell death ligand 1 (PD-L1), and poorer prognosis. In aHCC patients, cancer cells and stellate cells in TME appeared to express high level of PD-L1, which should suppress activities of antitumor immune cells. In this context, the combination of PD-1/PD-L1 inhibitors and other therapeutic modalities are tried to treat aHCC. The chimeric antigen receptor (CAR) T cell therapy is a potent way of eradicating cells expressing targetable antigens. Removal of PD-L1 expressing cells in the aHCC TME will improve the clinical outcomes of current therapies against aHCC. In the present study, we developed an autologous anti-PD-L1 CAR T strategy and show high efficacy of tumor suppression using an HCC xenograft model. [Result] We constructed second generation CAR lentiviral vector expressing an unique anti-PD-L1 scFv manifesting moderated affinity. The scFv was specifically generated by phage display technique using human B cell-derived naïve cDNA library, which targets PD-L1 with intermediate affinity in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with CD3 zeta chain. The effector CAR T, named VPC1, cells specifically recognized PD-L1 expressing HCC cells and exhibited rapid and robust cytotoxic activity in 2D and 3D culture experiments. In an HCC xenograft NSG mouse model, VPC1 CAR T cells significantly suppressed the tumor growth. More importantly, VPC-1-treated animals have well tolerated the CAR T therapy with no significant adverse manifestations during one month observation period and exhibited gradual weight gains since 3 days after CAR T cell injection. [Conclusion] We hereby report that a new autologous PD-L1 CAR-T therapeutic is capable of eradicating solid tumors expressing PD-L1, which would significantly potentiate efficacies of current conventional and immunotherapeutic strategies. Citation Format: Kyung-Sik Lee, Hye-Seong Park, Guk-Yeol Park, Gi-Chan Lee, Eun-Ji Choi, Jae-in Yu, Seung-Joo Yang, Mihwa Kim, Yoonjoo Choi, Je-Jung Lee, Joon Haeng Rhee, Bum Chan Park, Jae Eun Park. Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6189.
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Du, Xin-Jun, Yi-Na Wu, Wei-Wei Zhang, Feng Dong, and Shuo Wang. "Construction and quality examination of murine naive T7 phage display antibody library." Food and Agricultural Immunology 21, no. 1 (March 2010): 81–90. http://dx.doi.org/10.1080/09540100903414106.
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Liu, Hong, Li Long, Shon Green, Lucas H. Horan, Bryan Zimdahl, and Cheng Liu. "Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models." Blood 128, no. 22 (December 2, 2016): 3354. http://dx.doi.org/10.1182/blood.v128.22.3354.3354.
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Abstract Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies. Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison. When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies. When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10. While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2). Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. Figure 1 Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 1. Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 2 Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Figure 2. Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. / (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.
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Bu, Dexiu, Paul Bennett, Nathaniel Barton, Laura Bradshaw, Maria Pinon-Ortiz, Xiangen Li, Poonam Vaidya, et al. "Identification and Development of PHE885: A Novel and Highly Potent Fully Human Anti-BCMA CAR-T Manufactured with a Novel T-Charge TM Platform for the Treatment of Multiple Myeloma." Blood 138, Supplement 1 (November 5, 2021): 2770. http://dx.doi.org/10.1182/blood-2021-148390.
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Abstract Background : Chimeric antigen receptor T cell (CAR-T) therapies for multiple myeloma (MM) targeting B cell maturation antigen (BCMA) have demonstrated promising efficacy; however, many patients with relapsed/refractory (r/r) MM will ultimately relapse. To improve on existing BCMA CAR-Ts, two critical attributes of the CAR-T product were investigated: 1) the potency of the BCMA CAR construct, and 2) a rapid manufacturing process that would both preserve the stemness of T cells to ensure longer duration of response and provide timely access for patients with rapidly progressing, aggressive disease. Through extensive panning and discriminating CAR-T functional assays to assess performance, we have identified a superior anti-BCMA CAR construct that when combined with an innovative T-Charge™ manufacturing platform, produces a highly potent product. Methods and Results : We have developed a novel anti-BCMA CAR-T product for the treatment of MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) targeting BCMA, fused to the CD8α hinge and transmembrane domains, followed by CD137 (4-1BB) co-stimulatory and CD3ζ chain signaling domains. Selection of our development candidate was based on a screening of seventeen anti-BCMA CARs, each comprising a distinct scFv derived from phage display libraries and hybridoma with a wide range of affinities and epitopes. One candidate clone from the fully human B cell library was identified after rigorous assessment of transduction efficiency, CAR expression, antigen specificity/selectivity and CAR-T cell function against a MM cell line, both in vitro and in vivo. This scFv also demonstrates high specificity to human BCMA by Retrogenix platform using a commercial human plasma membrane protein array assay. CAR expression and functionality of the CAR constructs were compared in the CAR-T cells generated with two different cell processes: a traditional manufacturing (TM) in which CAR-T cells are expanded in vitro for 9-10 days and a novel T-Charge TM process, which is an expansionless CAR-T manufacturing process that takes <2days to generate functional CAR-Ts. PHE885 generated with the novel T-Charge TM manufacturing platform, retains naïve/ stem cell memory T cell (T scm) (CD45RO -/CCR7 +) while the traditionally manufactured cell product carrying the same scFv (TM_PHE885) mainly contains central-memory T cells (CD45RO +/CCR7 +). In addition to its unique phenotype, PHE885 secretes up to 25 fold more target specific IL-2 and ~7 fold more IFN gamma in vitro, when comparing to the TM products either using the same lentiviral vector (TM_PHE885) or a clinically validated anti-BCMA vector (MCM998). In an immunodeficient NOD-scid IL2R gamma null (NSG) mouse model of MM, PHE885 induced tumor regression at a very low dose in a dose-dependent manner, and was up to 5 fold more efficacious in eradicating tumors compared to the two TM products. This enhanced efficacy was accompanied by higher cellular expansion in vivo (up to 3 fold higher C max and AUC 0-21d). The ability of PHE885 to control the tumor at lower doses with a greater expansion profile confirms the robustness and potency of the PHE885 cells. Furthermore, unlike the TM products, PHE885 induced earlier graft-versus-host disease at medium and high tested doses in this mouse model, suggesting the stemness of the product is most likely the driver of stronger T cell expansion. Conclusions : PHE885 was discovered and designed to enhance potency and drive persistence of CAR-T through the combination of a novel CAR construct carrying a fully human anti-BCMA scFv fused to 4-1BB/CD3ζ signaling domains and a novel T-Charge TM manufacturing platform, which enables rapid and reliable patient access. The novel T-Charge TM platform allows PHE885 to preserve a significantly higher proportion of naïve/ T scm cells, enabling PHE885 to effectively engraft, expand and reject tumors at a dose of 5 fold lower than TM CAR-T cells. Based on these results, a Phase 1, open-label trial assessing PHE885 in patients with r/r MM (NCT04318327) was initiated. Initial data from the dose escalation portion of the Phase 1 study will be presented separately. Figure 1 Figure 1. Disclosures Bu: Novartis: Current Employment, Patents & Royalties: Co-inventor on patent applications. Bennett: BMS: Current Employment; Novartis: Ended employment in the past 24 months, Other: PB was a full-time employee of Novartis at the time of the study. Barton: BMS: Current Employment; Novartis: Other: NB was a full-time employee of Novartis at the time of the study. Bradshaw: Novartis: Other: LB was a full-time employee of Novartis at the time of the study; AstraZeneca: Current Employment. Pinon-Ortiz: NIBR: Current Employment, Current holder of stock options in a privately-held company. Li: Novartis: Current Employment. Vaidya: NIBR: Current Employment. Zhu: Novartis: Current equity holder in publicly-traded company; NIBR: Current Employment. Mansfield: Novartis: Current Employment. Granda: Novartis: Current Employment, Patents & Royalties: No royalties as company-held patents. Guimaraes: Novartis: Current Employment, Patents & Royalties: Co-inventor on patent applications. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.
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PELSERS, Maurice M. A. L., Jan T. LUTGERINK, Frans A. van NIEUWENHOVEN, Narendra N. TANDON, Ger J. van der VUSSE, Jan-Willem ARENDS, Hennie R. HOOGENBOOM, and Jan F. C. GLATZ. "A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes." Biochemical Journal 337, no. 3 (January 25, 1999): 407–14. http://dx.doi.org/10.1042/bj3370407.
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The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (< 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P< 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.
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Khan, Israr, Faiza Ahmed, Nazma Hanif, Mobeen Zaka Haider, Farhan Khalid, Sakshi Mishra, Radhika Garimella, et al. "Role of capmatinib in MET exon 14-mutated advanced non-small cell lung cancer (NSCLC): A systematic review." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e21150-e21150. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21150.
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e21150 Background: Capmatinib is a selective inhibitor of MET receptor that got accelerated FDA approval in May 2020 to treat NSCLC with MET ex 14 mutation (METex14). Here, we proclaim the first review of capmatinib efficacy and safety profile in METex14 positive NSCLC patients. Methods: A systematic literature search was conducted using PubMed, Cochrane Library, Clinicaltrials.gov, and Google Scholar. We compiled and analyzed the original studies evaluating the clinical response of capmatinib in MET ex14 mutated advanced NSCLC. Results: The most recent results (as of January 6, 2020) of the GEOMETRY Mono-1 phase 2 study showed an overall response rate (ORR) of 41% (69/97; 95% CI, 29-53) with 41% partial response (PR), and 36% stable disease (SD) in patients who previously received 1-2 prior therapy lines (cohort 4). While the ORR, PR, and SD were 68% ( n = 29/97; 95% CI, 48-84), 64%, and 25%, respectively in treatment naïve patients (cohort 5b). Median progression-free survival (mPFS) and duration of response (DOR) were 5.4 months (mon) and 9.7 mon (95%CI, 5.6-13.0) in cohort 4 vs. 12.4 mon and 12.6 mon (CI, 5.6- not estimated), respectively in cohort 5b. Moreover, disease control was shown in n = 54/69 patients in cohort 4 (95% CI 78 (97-87) and n = 27/28 patients (95% CI 96 (82-100). A post-hoc analysis of 19 patients out of 69 who were pre-treated with immunotherapy demonstrated an ORR of 57.9% (n = 11/19; 95%CI 33.5-79.7) with capmatinib. In contrast, treatment-naive had an ORR of 34% (n = 17/50; 95%CI 21.2-48.8) as per the investigator. Median PFS was 3.29 mon noted on prior therapy. In the phase 1 retrospective analysis reported by Choi et al., ORR was 50% with a median duration of 16.1 (5.3-36.4) mon. Among 45 Japanese pts with MET ex 14 mutated or MET amplified status, the ORR was 36.4% (95% CI 10.9%-69.2%) with a PFS of 4.70 mon in the MET ex 14 mutated groups who had received a second or third line of therapy. Most commonly reported adverse events were peripheral edema, nausea, vomiting, and elevated creatinine across all studies and were mostly grade 1-2. Conclusions: Our results showed that capmatinib has promising anti-tumor activity in patients with NSCLC harboring MET exon 14 skipping mutation. The efficacy and tolerability profile of capmatinib is remarkable, particularly in treatment-naïve patients. Although the GEOMETRY Mono-1 trial is still ongoing, further clinical studies with long-term follow-up are needed.
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Turunen, Laura, Kristiina Takkinen, Hans Söderlund, and Timo Pulli. "Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries." Journal of Biomolecular Screening 14, no. 3 (February 11, 2009): 282–93. http://dx.doi.org/10.1177/1087057108330113.
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Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. ( Journal of Biomolecular Screening 2009:282-293)
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Khatri, Ajay, Shweta Agrawal, and Jyotir M. Chatterjee. "Wheat Seed Classification: Utilizing Ensemble Machine Learning Approach." Scientific Programming 2022 (February 2, 2022): 1–9. http://dx.doi.org/10.1155/2022/2626868.
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Recognizing and authenticating wheat varieties is critical for quality evaluation in the grain supply chain, particularly for methods for seed inspection. Recognition and verification of grains are carried out manually through direct visual examination. Automatic categorization techniques based on machine learning and computer vision offered fast and high-throughput solutions. Even yet, categorization remains a complicated process at the varietal level. The paper utilized machine learning approaches for classifying wheat seeds. The seed classification is performed based on 7 physical features: area of wheat, perimeter of wheat, compactness, length of the kernel, width of the kernel, asymmetry coefficient, and kernel groove length. The dataset is collected from the UCI library and has 210 occurrences of wheat kernels. The dataset contains kernels from three wheat varieties Kama, Rosa, and Canadian, with 70 components chosen at random for the experiment. In the first phase, K-nearest neighbor, classification and regression tree, and Gaussian Naïve Bayes algorithms are implemented for classification. The results of these algorithms are compared with the ensemble approach of machine learning. The results reveal that accuracies calculated for KNN, decision, and Naïve Bayes classifiers are 92%, 94%, and 92%, respectively. The highest accuracy of 95% is achieved through the ensemble classifier in which decision is made based on hard voting.
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Willats, William G. T., Philip M. Gilmartin, Jorn Dalgaard Mikkelsen, and J. Paul Knox. "Cell wall antibodies without immunization: generation and use of de-esterified homogalacturonan block-specific antibodies from a naive phage display library." Plant Journal 18, no. 1 (April 1999): 57–65. http://dx.doi.org/10.1046/j.1365-313x.1999.00427.x.
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Hao, Jia, Yihang Li, Jingxuan Wang, Chongxin Xu, Meijing Gao, Wei Chen, Xiao Zhang, Xiaodan Hu, Yuan Liu, and Xianjin Liu. "Screening and activity identification of an anti-idiotype nanobody for Bt Cry1F toxin from the camelid naive antibody phage display library." Food and Agricultural Immunology 31, no. 1 (December 1, 2019): 1–16. http://dx.doi.org/10.1080/09540105.2019.1691156.
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Zarei, Bahareh, Zahra Javidan, Elnaz Fatemi, Fatemeh Rahimi Jamnani, Shohreh Khatami, and Vahid Khalaj. "Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library." DARU Journal of Pharmaceutical Sciences 28, no. 1 (March 19, 2020): 221–35. http://dx.doi.org/10.1007/s40199-020-00334-z.
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Carzaniga, Raffaella, Daniela Fiocco, Paul Bowyer, and Richard J. O'Connell. "Localization of Melanin in Conidia of Alternaria alternata Using Phage Display Antibodies." Molecular Plant-Microbe Interactions® 15, no. 3 (March 2002): 216–24. http://dx.doi.org/10.1094/mpmi.2002.15.3.216.
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Melanins derived from 1,8-dihydroxynaphthalene (DHN) are important for the pathogenicity and survival of fungi causing disease in both plants and animals. However, precise information on their location within fungal cell walls is lacking. To obtain antibodies for the immunocytochemical localization of melanin, 83 phage antibodies binding to 1,8-DHN were selected from a naive semisynthetic single-chain Fv (scFv) phage display library. Sequence analysis of the heavy chain binding domains of 17 antibodies showed a high frequency of positively charged amino acids. One antibody, designated M1, was characterized in detail. M1 bound specifically to 1,8-DHN in competitive inhibition enzyme-linked immunosorbent assays, showing no cross-reaction with nine structurally related phenolic compounds. Epitope recognition required two hydroxyl groups in a 1,8 configuration. M1 also bound to naturally occurring melanin isolated from mycelia of Alternaria alternata, suggesting that epitopes remain accessible in polymerized melanin. Transmission electron microscopy-immunogold labeling, using M1 in the form of soluble scFv fragments, showed that melanin was located in the septa and outer (primary) walls of wild-type A. alternata conidia, but not those of an albino mutant, AKT88-1. The M1 antibody provides a new tool for detecting melanized pathogens in plant and animal tissues and for precisely mapping the distribution of the polymer within spores, appressoria, and hyphae.
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Ciccarese, Chiara, Roberto Iacovelli, Emilio Bria, Giovanni Schinzari, Ernesto Rossi, Serena Astore, Maria Antonella Cannella, et al. "Efficacy of VEGFR-TKI plus immune checkpoint inhibitor (ICI) in metastatic renal cell carcinoma (mRCC) patients with favorable IMDC prognosis." Journal of Clinical Oncology 39, no. 6_suppl (February 20, 2021): 318. http://dx.doi.org/10.1200/jco.2021.39.6_suppl.318.
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318 Background: Combinations of a PD-1/PD-L1 immune checkpoint inhibitor (ICI) with a VEGFR-TKI as front-line/treatment-naïve therapy significantly improve the outcome of metastatic renal cell carcinoma (mRCC) patients. The benefit of these combinations is well evident in IMDC intermediate- and poor-risk population, while it is unclear in the subgroup of mRCC patients with favorable prognosis. We performed a meta-analysis with the aim to evaluate whether the addition of ICIs to VEGFR-TKIs is able to improve the outcome compared to VEGFR-TKIs alone in mRCC patients with favorable IMDC prognosis. Methods: This meta-analysis searched MEDLINE/PubMed, the Cochrane Library and ASCO Meeting abstracts for phase II or III randomized clinical trials (RCTs) testing the combination of VEGFR-TKI+ICI in mRCC. Data extraction was conducted according to the PRISMA statement. The hazard ratios (HRs) for PFS and OS with the relative 95% CIs were extracted from each study. Summary HRs was calculated using random- or fixed-effects models, depending on the heterogeneity of the included studies. Results: Three RCTs were selected for the final analysis, with a total of 605 patients (306 treated with VEGFR-TKI+ICI combinations and 299 who received sunitinib in the control arms). The combination of VEGFR-TKI+ICI improved PFS compared to sunitinib, with a 30% reduction of the risk of progression (fixed-effect, HR=0.70; p = 0.003). However, VEGFR-TKI+ICI combinations did not significantly prolong OS (fixed-effect; HR = 0.94; 95% CI 0.62–1.43; p = 0.77). Conclusions: Our analysis demonstrates a PFS benefit without an OS advantage for VEGFR-TKI+ICI combinations as first-line therapy for mRCC patients with favourable prognosis according to IMDC. Longer follow-up is required to definitely confirm the best therapy for treatment-naïve mRCC patients with favorable prognosis. [Table: see text]
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Buchwald, Ulrike K., Andrew Lees, Michael Steinitz, and Liise-anne Pirofski. "A Peptide Mimotope of Type 8 Pneumococcal Capsular Polysaccharide Induces a Protective Immune Response in Mice." Infection and Immunity 73, no. 1 (January 2005): 325–33. http://dx.doi.org/10.1128/iai.73.1.325-333.2005.
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ABSTRACT Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.
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Ju, Man-Seok, Hye-Mi Ahn, Seong-Gu Han, Sanghwan Ko, Jung-Hyun Na, Migyeong Jo, Chung Su Lim, et al. "A human antibody against human endothelin receptor type A that exhibits antitumor potency." Experimental & Molecular Medicine 53, no. 9 (September 2021): 1437–48. http://dx.doi.org/10.1038/s12276-021-00678-9.
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AbstractEndothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.
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Weiner, Adam C., Andrew McPherson, and Sohrab P. Shah. "Abstract 2128: Single-cell replication dynamics in genomically unstable cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2128. http://dx.doi.org/10.1158/1538-7445.am2022-2128.
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Abstract Background: Single-cell whole genome sequencing (scWGS) methods such as direct library preparation (DLP) provide amplification-free capture of cells in all cycle phases and have enabled rich interrogation into the cell to cell genomic diversity of cancer genomes. Previous DLP-driven clonal evolution studies removed S-phase cells as replicated loci confounded phylogenetic inference from somatic copy number aberrations (CNAs), leaving single-cell replication timing (scRT) as an unstudied property. We thus developed a method that assigned S-phase cells to phylogenetic clones and used such assignments to infer scRT profiles in aneuploid cell lines and tumors. We applied this method to determine the relative proliferation rate between clones and obtain a single-cell picture of DNA replication fork progression for genomically unstable cancers. Methods: S-phase cells were assigned to phylogenetic clones based on copy number profile similarity and subsequently scRT profiles were inferred by normalizing out the effects of clonal, subclonal, and cell-specific CNAs before binarizing each genomic bin as replicated or unreplicated. scRT heterogeneity was quantified via a summary statistic representing the aggregate difference between expected and observed replication times (T-width). The scRT profiles were grouped into clone- and sample-level pseudobulks (aggregations of single cells) to determine genomic regions with differential replication timing. We then assessed the ability of S-phase enrichment to predict clonal expansions on time-series patient-derived xenografts of HER2+ and triple negative breast cancers that were either treatment-naive or exposed to cisplatin. In addition, we studied genetically engineered cell lines with inactivated DNA damage response genes (TP53, BRCA1, BRCA2) and primary high-grade serous ovarian cancer samples to quantify scRT heterogeneity at different states of genomic instability. Results: We analyzed 15 datasets containing over 25,000 cells with ~1/3 of cells in S-phase. Time-series datasets revealed that S-phase enriched clones were highly proliferative in the treatment-naive setting and were most sensitive to cisplatin. T-width values calculated from inferred scRT profiles increased as a function of genomic instability, with the degree of heterogeneity varied between genomic regions. Clone-level pseudobulk replication timing profiles revealed that whole-chromosome aneuploidies were more likely to have unperturbed replication timing compared to complex structural variants such as translocations, inversions, and micronuclei. Conclusions: Our time-series results show that S-phase cells enable the prediction of clone-specific proliferation and chemosensitivity using scWGS data from a single time point. The quantification of scRT heterogeneity across our collection of data implicates dysfunctional DNA replication as both driver and consequence of genomic instability. Citation Format: Adam C. Weiner, Andrew McPherson, Sohrab P. Shah. Single-cell replication dynamics in genomically unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2128.
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Cirino, Nick M., Daniele Sblattero, David Allen, Scott R. Peterson, James D. Marks, Paul J. Jackson, Andrew Bradbury, and Bruce E. Lehnert. "Disruption of Anthrax Toxin Binding with the Use of Human Antibodies and Competitive Inhibitors." Infection and Immunity 67, no. 6 (June 1, 1999): 2957–63. http://dx.doi.org/10.1128/iai.67.6.2957-2963.1999.
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ABSTRACT The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.
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Rink, L., Y. Skorobogatko, A. Kossenkov, M. G. Belinsky, T. F. Pajak, M. von Mehren, M. F. Ochs, B. Eisenberg, and A. K. Godwin. "Correlation of gastrointestinal stromal tumor (GIST) gene expression signatures and response to imatinib mesylate in the Radiation Therapy Oncology Group phase II clinical trial S-0132." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 10533. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.10533.
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10533 Background: Despite the known efficacy of imatinib mesylate (IM) in metastatic GIST, both primary and secondary drug resistance remain a clinical problem. Therefore, future management of GIST may benefit from further molecular characterization associated with IM treatment. Methods: A recently completed prospective phase II trial of neoadjuvant/adjuvant IM treatment for advanced primary and recurrent operable GISTs included gene expression profiling using oligonucleotide microarrays on tumor samples obtained before and after neoadjuvant IM therapy for 8 to 12 weeks. Fifty-two analyzable patients were classified according to changes in tumor size after IM based on CT scan measurements. A response was defined as >25% tumor reduction. Gene profiling data were then evaluated with SAM analysis (FDR = 10%) to identify differentially expressed genes (in pre-treatment GIST samples). In addition, a custom siRNA library was designed targeting these genes and a mid-throughput siRNA synthetic lethal screening approach was used to evaluate the ability of each to sensitize GIST cells to IM. Results: Thirty-eight genes were expressed at significantly lower levels in the pre-treatment samples of tumors that rapidly responded to IM. Eighteen of these genes encoded KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. Importantly, ten KRAB-ZNF genes mapped to a single locus on chromosome 19p, and a subset of these predicted likely response to IM-based therapy in a naïve panel of GISTs. Further evaluation revealed that modifying in vitroexpression of genes via RNAi approaches within this predictive response signature enhanced the sensitivity of GIST cells to IM. Conclusions: Using clinical samples from a prospective neoadjuvant/adjuvant phase II trial we have identified a genetic signature which includes KRAB-ZNF 91 subfamily members that may be both predictive of and functionally associated with likely rapid response to short-term IM treatment and which may provide a prognostic biomarker identifying patients who will benefit from it. [Table: see text]
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Elaidi, Reza, Letuan Phan, Delphine Borchiellini, Philippe Barthelemy, Alain Ravaud, Stéphane Oudard, and Yann Vano. "Comparative Efficacy of First-Line Immune-Based Combination Therapies in Metastatic Renal Cell Carcinoma: A Systematic Review and Network Meta-Analysis." Cancers 12, no. 6 (June 24, 2020): 1673. http://dx.doi.org/10.3390/cancers12061673.
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Three drug combinations, ipilimumab-nivolumab (Ipi-Nivo), pembrolizumab-axitinib (Pembro-Axi), and avelumab-axitinib (Ave-Axi), have received regulatory approval in the USA and Europe for the treatment of metastatic renal cell carcinoma with clear cell component (mRCC). However, no head-to-head comparison data are available to identify the best option. Therefore, we aimed to compare these new treatments in a first-line setting. We conducted a systematic search in PubMed, the Cochrane Library, and clinicaltrials.gov for any randomized controlled trials of treatment-naïve patients with mRCC, from January 2015 to October 2019. The process was performed according to PRISMA guidelines. We performed a Bayesian network meta-analysis with two different approaches, a contrast-based model comparing HRs and ORs between studies and arm-based using parametric modeling. The outcomes for the analysis were overall survival, progression-free survival (PFS), and objective response rate. Our search identified 3 published phase 3 randomized clinical trials (2835 patients). In the contrast-based model, Ave-Axi (SUCRA = 83%) and Pembro-Axi (SUCRA = 80%) exhibited the best ranking probabilities for PFS. For overall survival (OS), Pembro-Axi (SUCRA = 96%) was the most preferable option against Ave-Axi and Ipi-Nivo. Objective response rate analysis showed Ave-Axi as the best (SUCRA: 94%) and Pembro-Axi as the second best option. In the parametric models, the risk of progression was comparable for Ave-Axi and Ipi-Nivo, whereas Pembro-Axi exhibited a lower risk during the first 6 months of treatment and a higher risk afterwards. Furthermore, Pembro-Axi exhibited a net advantage in terms of OS over the two other regimens, while Ave-Axi was the least preferable option. Overall evidence suggests that pembrolizumab plus axitinib seems to have a slight advantage over the other two combinations.
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Chan, Wee Lee William, Vanessa CL Chong, Ian JY Wee, Anand Jeyasekharan, Michelle Poon, Hian Li Esther Chan, Joanne Lee, et al. "Evaluating Front Line Treatment Regimens for Waldenstrom Macroglobulinaemia: A Systematic Review and Meta-Analysis." Blood 138, Supplement 1 (November 5, 2021): 1358. http://dx.doi.org/10.1182/blood-2021-150678.
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Abstract Background Waldenstrom macroglobulinemia (WM) is a rare haematological malignancy characterised by infiltration of the bone marrow by clonal lymphoplasmocytic cells and serum monoclonal IgM gammopathy. Rituximab based chemo-immunotherapy has long been the backbone of WM treatment. Bendamustine rituximab (BR), cyclophosphamide, dexamethasone rituximab (CRD) and bortezomib dexamethasone rituximab (BDR) are currently considered frontline options. The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib in combination with rituximab (IR) has recently shown efficacy in treatment naïve WM and has been approved for use in this setting. With the expanding number of treatment options and absence of randomised trials (RCTs) comparing IR with chemo immunotherapy, there is uncertainty as to the optimal frontline treatment for WM. Methods The aim of this systematic review was to compare the efficacy and safety of rituximab-based chemoimmunotherapy combinations and IR, in treatment naïve WM. Using a pre-specified protocol in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA), MEDLINE, Embase, BIOSIS and Cochrane Library databases were searched for articles published from inception of each database to present. We included trials studying newly diagnosed WM patients treated with rituximab-based chemo-immunotherapy or BTKi based regimens. Primary outcome measures were progression free and overall survival (PFS and OS) while secondary outcomes included response rates and adverse events. A meta-analysis of proportions (expressed as a percentage), with their 95% CI were calculated. Results Of the 753 unique records identified, 8 single arm studies, one phase II non-randomised non inferiority study and 3 Phase III RCTs met the inclusion criteria. The sample size ranged from 23 to 261. PFS at 1- and 2-years were higher in BR(91%, 87%) and IR (93%, 82%), compared to BDR (87.5%, 66.8%) and CRD (82%, 64%) . Combined complete response (CR) and very good partial response (VGPR) rates were also superior with BR (35%) and IR ( 26%) compared to BDR (9%), Bortezomib- Rituximab (8% ) and CRD ( 7%) (Figure 1). The bortezomib bendamustine rituximab ( BBR) regimen was evaluated in only one study but showed an impressive CR/VGPR rate of 47% and a PFS at 3 years of 75%. Bleeding and atrial fibrillation were more common with IR, while neuropathy was more prevalent with bortezomib based regimens. Conclusion We present the first systematic review and meta-analysis evaluating frontline treatment options for WM in the BTKi era. Our findings suggest that bendamustine-based regimens may be superior in terms of depth of response and PFS. Longer follow up of the IR treated patients on the INNOVATE trial is however required to perform a definitive comparison of the regimens. The differential efficacy of these treatments in genomic subtypes of WM (based on MYD88 and CXCR4 mutational status) is an important question to be answered in future trials. While chemo-immunotherapy remains a potent, fixed duration option for newly diagnosed WM, BTKi based regimens are a valid alternative for patients who prefer an oral treatment or who are unfit for cytotoxic therapy. With the expanding number of treatment options for WM, RCTs comparing BTKi/immunotherapy with chemo-immunotherapy regimens are called for. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Sumanasuriya, Semini, George Seed, Niven Mehra, Rossitza Christova, Lorna Pope, Jane Goodall, Penelope Flohr, et al. "Cell-free DNA as a biomarker for taxane treatment in advanced prostate cancer." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 5070. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.5070.
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5070 Background: Antitumor efficacy of chemotherapeutic drugs docetaxel and cabazitaxel against hormone-sensitive and castration-resistant prostate cancer (CRPC) is well recognized. The identification and validation of minimally invasive biomarkers of taxane response remain of paramount importance. Methods: Serial pre-, during and post-treatment plasma samples were collected prospectively from two large Phase III clinical trials, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Chemotherapy-naïve patients (pts) were treated with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2) in FIRSTANA, and pts previously treated with docetaxel received cabazitaxel (20 or 25 mg/m2) as second-line chemotherapy in PROSELICA. Plasma cell-free DNA (cfDNA) was extracted, low-pass whole genome sequencing (lp-WGS) libraries were prepared using the QIAGEN QiaSeq FX DNA library kit, and samples sequenced on the Illumina NovaSeq 6000. Targeted next-generation sequencing (NGS) on a custom-made AmpliSeq panel of 30 genes, pre-selected for putative roles in taxane resistance, was also performed. lp-WGS copy number (CN) profiles were generated using ichorCNA (v0.1.0) and targeted NGS variant calls derived using IonReporter software. Results: Overall, lp-WGS data was generated from 265 samples (99 pts; 51 treated on the FIRSTANA study, 48 treated on the PROSELICA study), acquired at three time-points (pre-, during and at progression). Average cfDNA input was 10 ng and mean coverage achieved was ~2X (SD 1.2X). We observed changes in lp-WGS tumor purity over time as a result of therapy; samples from non-responding pts exhibited significantly higher tumor purity values post-treatment compared with samples from responding pts (p = 0.02). Targeted NGS results were available from 294 pts (153 from FIRSTANA, 141 from PROSELICA), and changes in tumor purity were also associated with treatment response (p = 0.04). CN frequency of key CRPC genes was similar to previously reported datasets; several aberrant loci associated with response to taxane therapy. Conclusions: cfDNA lp-WGS may have clinical utility in the management of lethal prostate cancer. Funding: Sanofi. Clinical trial information: NCT01308580, NCT01308567.
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Essa, Hasanain Ali Al, and Wesam S. Bhaya. "Network Attacks Detection Depend on Majority Voting – Weighted Average for Feature Selection and Various Machine Learning Approaches." Webology 19, no. 1 (January 20, 2022): 2054–66. http://dx.doi.org/10.14704/web/v19i1/web19139.
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Due to the enormous growth in Internet usage and computer networks in recent years, new risks and challenges have arisen to network security. Among lots of security problems, network attack is a significant one. For instance, Distributed Denial of Service (DDoS) attacks have become appealing to intruders, and these have presented destructive threats to network infrastructures. Thus, Intrusion Detection Systems (IDSs) and Machine Learning (ML) approaches play a key role to detect such attacks effectively and efficiently. An essential part of several classification issues is the feature selection phase because to detect DDoS attacks depends on how one selects the minimal and relevant features in the network traffics. Unlike recent studies, in this work, a real-life SNMP-MIB dataset is used, as well as, we suggest an Ensemble-Weighted average approach (EnWaFS) that excludes the irrelevant features. An EnWaFS approach consists of two methods, first, Ensemble features by using a majority-voting method that mixed the outcomes of three feature selection approaches, second, a weighted average method that gives one weight for each feature and diminishes also the number of attributes. To evaluate an EnWaFS approach, we have performed four Machine Learning classifiers Neural network (Multi-Layer Perceptron), Vector Support Machine (SVM), Naïve Bayes (NB), and Random Forest (RF) utilizing the optimal set of attributes. The results reveal that our EnWaFS approach can efficiently decrease the number of attributes from 34 to 12 and also, from four ML classifiers were used, the RF technique achieved better performance due to the accuracy, sensitivity (recall), F-1 measure, precision, true-positive-rate, and the false-positive-rate which is decreased.