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İncir İ, Kaplan Ö. Escherichia coli in the production of biopharmaceuticals. Biotechnol Appl Biochem 2025; 72:528-541. [PMID: 39245907 PMCID: PMC11975263 DOI: 10.1002/bab.2664] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Accepted: 08/23/2024] [Indexed: 09/10/2024]
Abstract
Escherichia coli has shouldered a massive workload with the discovery of recombinant DNA technology. A new era began in the biopharmaceutical industry with the production of insulin, the first recombinant protein, in E. coli and its use in treating diabetes. After insulin, many biopharmaceuticals produced from E. coli have been approved by the US Food and Drug Administration and the European Medicines Agency to treat various human diseases. Although E. coli has some disadvantages, such as lack of post-translational modifications and toxicity, it is an important host with advantages such as being a well-known bacterium in recombinant protein production, cheap, simple production system, and high yield. This study examined biopharmaceuticals produced and approved in E. coli under the headings of peptides, hormones, enzymes, fusion proteins, antibody fragments, vaccines, and other pharmaceuticals. The topics on which these biopharmaceuticals were approved for treating human diseases, when and by which company they were produced, and their use and development in the field are included.
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Affiliation(s)
- İbrahim İncir
- Kazım Karabekir Vocational School, Department of Medical Services and Techniques, Environmental Health ProgramKaramanoğlu Mehmetbey UniversityKaramanTurkey
| | - Özlem Kaplan
- Rafet Kayış Faculty of Engineering, Department of Genetics and BioengineeringAlanya Alaaddin Keykubat UniversityAntalyaTurkey
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2
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Kim D, Kim JS, Bai X, Zhang J, Park M, Lee U, Lee J, Bahn YS, Xu Y, Ha NC. Development of Miniprotein-Type Inhibitors of Biofilm Formation in Candida albicans and Candida auris. J Microbiol Biotechnol 2025; 35:e2411076. [PMID: 40016146 PMCID: PMC11896806 DOI: 10.4014/jmb.2411.11076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2024] [Revised: 12/26/2024] [Accepted: 12/31/2024] [Indexed: 03/01/2025]
Abstract
Candida auris is a pathogenic fungus associated with high-mortality infections and forms resilient biofilms on various surfaces. In this study, we introduced a novel antifungal strategy against C. auris by integrating an AI-powered protein design tool, ProteinMPNN, with classical molecular dynamics (MD) simulations to design artificial proteins from a miniprotein library. This combined approach accelerated and enhanced the design process, enabling the rapid development of effective miniprotein inhibitors specifically targeting C. auris biofilm formation. The miniproteins developed in this study exhibited potent inhibitory effects on C. auris biofilms, representing a significant advancement in antifungal therapy. Notably, the combined application of these miniproteins enhanced suppression of biofilm formation. These findings highlight not only the strong therapeutic potential of these designed miniproteins but also the power of combining AI-driven protein design with MD simulations to advance biomedical research.
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Affiliation(s)
- Doyeon Kim
- Research Institute of Agriculture and Life Sciences, Department of Agricultural Biotechnology, CALS, Seoul National University, Seoul 08826, Republic of Korea
| | - Ji-Seok Kim
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
| | - Xue Bai
- Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, China
| | - Jie Zhang
- Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, China
| | - Minho Park
- Research Institute of Agriculture and Life Sciences, Department of Agricultural Biotechnology, CALS, Seoul National University, Seoul 08826, Republic of Korea
| | - Ungyu Lee
- Research Institute of Agriculture and Life Sciences, Department of Agricultural Biotechnology, CALS, Seoul National University, Seoul 08826, Republic of Korea
| | - Jinwook Lee
- Hanmi Pharmaceutical Co., Ltd., Seoul, Republic of Korea
| | - Yong-Sun Bahn
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
| | - Yongbin Xu
- Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, China
| | - Nam-Chul Ha
- Research Institute of Agriculture and Life Sciences, Department of Agricultural Biotechnology, CALS, Seoul National University, Seoul 08826, Republic of Korea
- Center for Food and Bioconvergence, Department of Agricultural Biotechnology, Interdisciplinary Programs in Agricultural Genomics, CALS, Seoul National University, Seoul 08826, Republic of Korea
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3
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Hallaji M, Allahyari M, Teimoori-Toolabi L, Yasami-Khiabani S, Golkar M, Fard-Esfahani P. Targeted cancer treatment using a novel EGFR-specific Fc-fusion peptide based on GE11 peptide. Sci Rep 2025; 15:5107. [PMID: 39934226 PMCID: PMC11814073 DOI: 10.1038/s41598-025-89143-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Accepted: 02/03/2025] [Indexed: 02/13/2025] Open
Abstract
Fc-fusion peptides, also known as peptibodies, are a promising new category of targeted therapeutics that offer alternatives to monoclonal antibodies (mAbs) for cancer treatment. This study focuses on an Fc-fusion peptide consisting of the Fc region of IgG1 and an epidermal growth factor receptor (EGFR)-targeting peptide, GE11, which was identified using the phage display method and demonstrated high affinity for the receptor. The fusion peptide (FcIgG-GE11) was successfully expressed in Escherichia coli and purified using ion-exchange chromatography. Flow cytometry confirmed its specific binding to EGFR. Like Cetuximab, the FcIgG-GE11 peptibody exhibited effective, dose- and time-dependent growth inhibition of EGFR-overexpressing cancer cell lines. Additionally, the results showed that the FcIgG-GE11 peptibody induced cell death or cycle arrest in certain cancer cell lines, with varying responses depending on the cancer type. The results of In-Cell ELISA when comparing the effects of the FcIgG-GE11 peptibody to Cetuximab on Tyr 1173 phosphorylation were similar. In addition, the relative potency of the FcIgG-GE11 peptibody compared to Cetuximab was assessed using the MTT results by Slope Ratio Analysis. These findings suggest that FcIgG-GE11 peptibody can provide a specific and efficient tool for both targeting and treating cancer cells.
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Affiliation(s)
- Malihe Hallaji
- Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
| | - Mojgan Allahyari
- Recombinant Protein Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, Iran
| | - Ladan Teimoori-Toolabi
- Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | | | - Majid Golkar
- Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
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Binder U, Skerra A. Strategies for extending the half-life of biotherapeutics: successes and complications. Expert Opin Biol Ther 2025; 25:93-118. [PMID: 39663567 DOI: 10.1080/14712598.2024.2436094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Accepted: 11/26/2024] [Indexed: 12/13/2024]
Abstract
INTRODUCTION Engineering of the drug half-life in vivo has become an integral part of modern biopharmaceutical development due to the fact that many proteins/peptides with therapeutic potential are quickly cleared by kidney filtration after injection and, thus, circulate only a few hours in humans (or just minutes in mice). AREAS COVERED Looking at the growing list of clinically approved biologics that have been modified for prolonged activity, and also the plethora of such drugs under preclinical and clinical development, it is evident that not one solution fits all needs, owing to the vastly different structural features and functional properties of the pharmacologically active entities. This article provides an overview of established half-life extension strategies, as well as of emerging novel concepts for extending the in vivo stability of biologicals, and their pros and cons. EXPERT OPINION Beyond the classical and still dominating technologies for improving drug pharmacokinetics and bioavailability, Fc fusion and PEGylation, various innovative approaches that offer advantages in different respects have entered the clinical stage. While the Fc fusion partner may be gradually superseded by engineered albumin-binding domains, chemical PEGylation may be replaced by biodegradable recombinant amino-acid polymers like PASylation, thus also offering a purely biotechnological manufacturing route.
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Affiliation(s)
| | - Arne Skerra
- Lehrstuhl für Biologische Chemie, Technische Universität München, Freising, Germany
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Mondéjar-Parreño G, Sánchez-Pérez P, Cruz FM, Jalife J. Promising tools for future drug discovery and development in antiarrhythmic therapy. Pharmacol Rev 2025; 77:100013. [PMID: 39952687 DOI: 10.1124/pharmrev.124.001297] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 08/30/2024] [Accepted: 10/04/2024] [Indexed: 01/22/2025] Open
Abstract
Arrhythmia refers to irregularities in the rate and rhythm of the heart, with symptoms spanning from mild palpitations to life-threatening arrhythmias and sudden cardiac death. The complex molecular nature of arrhythmias complicates the selection of appropriate treatment. Current therapies involve the use of antiarrhythmic drugs (class I-IV) with limited efficacy and dangerous side effects and implantable pacemakers and cardioverter-defibrillators with hardware-related complications and inappropriate shocks. The number of novel antiarrhythmic drugs in the development pipeline has decreased substantially during the last decade and underscores uncertainties regarding future developments in this field. Consequently, arrhythmia treatment poses significant challenges, prompting the need for alternative approaches. Remarkably, innovative drug discovery and development technologies show promise in helping advance antiarrhythmic therapies. In this article, we review unique characteristics and the transformative potential of emerging technologies that offer unprecedented opportunities for transitioning from traditional antiarrhythmics to next-generation therapies. We assess stem cell technology, emphasizing the utility of innovative cell profiling using multiomics, high-throughput screening, and advanced computational modeling in developing treatments tailored precisely to individual genetic and physiological profiles. We offer insights into gene therapy, peptide, and peptibody approaches for drug delivery. We finally discuss potential strengths and weaknesses of such techniques in reducing adverse effects and enhancing overall treatment outcomes, leading to more effective, specific, and safer therapies. Altogether, this comprehensive overview introduces innovative avenues for personalized rhythm therapy, with particular emphasis on drug discovery, aiming to advance the arrhythmia treatment landscape and the prevention of sudden cardiac death. SIGNIFICANCE STATEMENT: Arrhythmias and sudden cardiac death account for 15%-20% of deaths worldwide. However, current antiarrhythmic therapies are ineffective and have dangerous side effects. Here, we review the field of arrhythmia treatment underscoring the slow progress in advancing the cardiac rhythm therapy pipeline and the uncertainties regarding evolution of this field. We provide information on how emerging technological and experimental tools can help accelerate progress and address the limitations of antiarrhythmic drug discovery.
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Affiliation(s)
| | | | | | - José Jalife
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; Department of Medicine, University of Michigan, Ann Arbor, Michigan; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.
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Khezri H, Mostafavi M, Dabirmanesh B, Khajeh K. Peptibodies: Bridging the gap between peptides and antibodies. Int J Biol Macromol 2024; 278:134718. [PMID: 39142490 DOI: 10.1016/j.ijbiomac.2024.134718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2024] [Revised: 08/04/2024] [Accepted: 08/11/2024] [Indexed: 08/16/2024]
Abstract
Peptides are a very critical class of pharmaceutical compounds that can control several signaling pathways and thereby affect many physiological and biochemical processes. Previous research suggests that both peptides and antibodies may serve as potent tools for research, diagnostics, vaccination, and therapeutics across diverse domains. The distinct attributes of peptides, like their profound tissue penetration, efficient cellular internalization, reduced immunogenicity, and adaptability to chemical modification, underscore their significance in biomedical applications. However, they also possess drawbacks such as lower affinity, poor absorption, low stability to proteolytic digestion, and rapid clearance. The advent of peptibodies is a significant advance that improves the limitations of both peptides and antibodies. Peptibodies, or Peptide-Fc fusions, represent a promising therapeutic modality comprising biologically active peptides fused to an Fc domain. The stability and efficacy of the peptide are enhanced by this fusion strategy, which overcomes some of the inherent limitations. Many peptibodies have been developed to treat conditions like cancer, diabetes, and lupus. Romiplostim and Dulaglutide are the only ones approved by the EMA and FDA, respectively. Given the growing significance of peptibodies in the pharmaceutical landscape, this investigation aims to explain key aspects encompassing the intrinsic properties of peptides, the intricacies of peptibody production, and their potential therapeutic applications.
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Affiliation(s)
- Hamidhossein Khezri
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mahdiyeh Mostafavi
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
| | - Bahareh Dabirmanesh
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
| | - Khosro Khajeh
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
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Darbandi A, Abdi M, Dashtbin S, Yaghoubi S, Sholeh M, Kouhsari E, Darbandi T, Ghanavati R, Taheri B. Antibody-Antibiotic Conjugates: A Comprehensive Review on Their Therapeutic Potentials Against BacterialInfections. J Clin Lab Anal 2024; 38:e25071. [PMID: 38867639 PMCID: PMC11211676 DOI: 10.1002/jcla.25071] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 04/09/2024] [Accepted: 05/09/2024] [Indexed: 06/14/2024] Open
Abstract
INTRODUCTION Antibodies are significant agents in the immune system and have proven to be effective in treating bacterial infections. With the advancement of antibody engineering in recent decades, antibody therapy has evolved widely. AIM This review aimed to investigate a new method as a therapeutic platform for the treatment of bacterial infections and explore the novel features of this method in conferring pathogen specificity to broad-spectrum antibiotics. MATERIAL AND METHODS A literature review was conducted addressing the following topics about antibody-antibiotic conjugates (AACs): (1) structure and mechanism of action; (2) clinical effectiveness; (3) advantages and disadvantages. RESULT Antibody conjugates are designed to build upon the progress made in the development of monoclonal antibodies for the treatment of diseases. Despite the growing emergence of antibiotic resistance among pathogenic bacteria worldwide, novel antimicrobials have not been sufficiently expanded to combat the global crisis of antibiotic resistance. A recently developed strategy for the treatment of infectious diseases is the use of AACs, which are specifically activated only in host cells. CONCLUSION A novel therapeutic AAC employs an antibody to deliver the antibiotic to the bacteria. The AACs can release potent antibacterial components that unconjugated forms may not exhibit with an appropriate therapeutic index. This review highlights how this science has guided the design principles of an impressive AAC and discusses how the AAC model promises to enhance the antibiotic effect against bacterial infections.
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Affiliation(s)
- Atieh Darbandi
- Molecular Microbiology Research CenterShahed UniversityTehranIran
| | - Milad Abdi
- Research Center of Tropical and Infectious DiseasesKerman University of Medical SciencesKermanIran
| | - Shirin Dashtbin
- Department of Microbiology, School of MedicineIran University of Medical SciencesTehranIran
| | - Sajad Yaghoubi
- Basic Sciences DepartmentNeyshabur University of Medical SciencesNeyshaburIran
| | - Mohammad Sholeh
- Department of BacteriologyPasteur Institute of IranTehranIran
| | - Ebrahim Kouhsari
- Laboratory Sciences Research CenterGolestan University of Medical SciencesGorganIran
| | - Talieh Darbandi
- Pharmaceutical Sciences BranchIslamic Azad University of Medical SciencesTehranIran
| | | | - Behrouz Taheri
- Department of Medical Biotechnology, School of MedicineAhvaz Jundishapur University of Medical SciencesAhvazIran
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Ng CL, Lim TS, Choong YS. Application of Computational Techniques in Antibody Fc-Fused Molecule Design for Therapeutics. Mol Biotechnol 2024; 66:568-581. [PMID: 37742298 DOI: 10.1007/s12033-023-00885-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Accepted: 08/23/2023] [Indexed: 09/26/2023]
Abstract
Since the advent of hybridoma technology in the year 1975, it took a decade to witness the first approved monoclonal antibody Orthoclone OKT39 (muromonab-CD3) in the year 1986. Since then, continuous strides have been made to engineer antibodies for specific desired effects. The engineering efforts were not confined to only the variable domains of the antibody but also included the fragment crystallizable (Fc) region that influences the immune response and serum half-life. Engineering of the Fc fragment would have a profound effect on the therapeutic dose, antibody-dependent cell-mediated cytotoxicity as well as antibody-dependent cellular phagocytosis. The integration of computational techniques into antibody engineering designs has allowed for the generation of testable hypotheses and guided the rational antibody design framework prior to further experimental evaluations. In this article, we discuss the recent works in the Fc-fused molecule design that involves computational techniques. We also summarize the usefulness of in silico techniques to aid Fc-fused molecule design and analysis for the therapeutics application.
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Affiliation(s)
- Chong Lee Ng
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden, Penang, Malaysia
| | - Theam Soon Lim
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden, Penang, Malaysia
| | - Yee Siew Choong
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden, Penang, Malaysia.
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Yassin MA, Al-Rasheed M, Al-Khaboori M, Marashi M, Osman H, Wali Y, Al Kindi S, Alsayegh F, Provan D. Thrombopoietin-receptor agonists for adult patients with immune thrombocytopenia: a narrative review and an approach for managing patients fasting intermittently. Front Cardiovasc Med 2023; 10:1260487. [PMID: 38162126 PMCID: PMC10755910 DOI: 10.3389/fcvm.2023.1260487] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Accepted: 11/17/2023] [Indexed: 01/03/2024] Open
Abstract
Introduction Thrombopoietin-receptor agonist (TPO-RAs) currently represent the state of art for treating immune thrombocytopenia. Their different molecular structures contribute to the difference in their pharmacodynamics and pharmacokinetics. This narrative review aims to provide an overview of the current TPO-RAs approved for primary immune thrombocytopenia (romiplostim, eltrombopag, avatrombopag) and the effect of intermittent fasting in adult patients receiving TPO-RAs. Areas covered Literature was searched with no limits on date or language, using various combinations of keywords. Data on the pharmacokinetics, pharmacodynamics, efficacy, and safety of TPO-RAs and the effect of intermittent fasting were summarized. Expert opinion Switching between TPO-RAs is a useful strategy to tackle some associated limitations. Romiplostim and avatrombopag have an advantage over eltrombopag as they do not require any dietary restrictions. In cases where romiplostim and avatrombopag are unavailable, patients should be educated on the appropriate administration, possible interactions, and dietary restrictions before initiating eltrombopag.
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Affiliation(s)
- Mohamed A. Yassin
- National Center for Cancer Care and Research, Hematology Section, Hamad Medical Corporation, Doha, Qatar
| | - Mona Al-Rasheed
- Hematology Unit, Department of Medicine, Al-Adan Hospital, Hadiya, Kuwait
| | | | - Mahmoud Marashi
- Dubai Academic Health Corporation, Dubai, United Arab Emirates
| | - Hani Osman
- Hematology-Oncology Department, Tawam Hospital, Abu Dhabi, United Arab Emirates
| | - Yasser Wali
- Department of Child Health, Sultan Qaboos University, Muscat, Oman
| | - Salam Al Kindi
- Department of Hematology, Sultan Qaboos University, Muscat, Oman
| | - Faisal Alsayegh
- Faculty of Medicine, Department of Medicine, Health Sciences Center, Kuwait University, Kuwait City, Kuwait
| | - Drew Provan
- Academic Haematology Unit, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, United Kingdom
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Rana S, Ughade S, Kumthekar R, Bhambure R. Chromatography assisted in-vitro refolding and purification of recombinant peptibody: Recombinant Romiplostim a case study. Int J Biol Macromol 2023; 249:126037. [PMID: 37516226 DOI: 10.1016/j.ijbiomac.2023.126037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 07/16/2023] [Accepted: 07/26/2023] [Indexed: 07/31/2023]
Abstract
In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.
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Affiliation(s)
- Sunil Rana
- Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Santosh Ughade
- Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Rupali Kumthekar
- Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Rahul Bhambure
- Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
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11
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Sargis T, Youn SW, Thakkar K, Naiche LA, Paik NY, Pajcini KV, Kitajewski JK. Notch1 and Notch4 core binding domain peptibodies exhibit distinct ligand-binding and anti-angiogenic properties. Angiogenesis 2023; 26:249-263. [PMID: 36376768 PMCID: PMC10119233 DOI: 10.1007/s10456-022-09861-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Accepted: 11/05/2022] [Indexed: 11/16/2022]
Abstract
The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.
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Affiliation(s)
- Timothy Sargis
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Seock-Won Youn
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Krishna Thakkar
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - L A Naiche
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Na Yoon Paik
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Kostandin V Pajcini
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, 60612, USA
- University of Illinois Cancer Center, University of Illinois Chicago, Chicago, IL, 60612, USA
| | - Jan K Kitajewski
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA.
- University of Illinois Cancer Center, University of Illinois Chicago, Chicago, IL, 60612, USA.
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12
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Chidipi B, Chang M, Cui M, Abou-Assali O, Reiser M, Pshenychnyi S, Logothetis DE, Teng MN, Noujaim SF. Bioengineered peptibodies as blockers of ion channels. Proc Natl Acad Sci U S A 2022; 119:e2212564119. [PMID: 36475947 PMCID: PMC9897444 DOI: 10.1073/pnas.2212564119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 10/25/2022] [Indexed: 12/12/2022] Open
Abstract
We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.
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Affiliation(s)
- Bojjibabu Chidipi
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL33612
| | - Mengmeng Chang
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL33612
| | - Meng Cui
- Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Center for Drug Discovery, Northeastern University, Boston, MA02115
| | - Obada Abou-Assali
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL33612
| | - Michelle Reiser
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL33612
| | - Sergii Pshenychnyi
- Chemistry for Life Processes Institute, Northwestern University, Evanston, IL60208
| | - Diomedes E. Logothetis
- Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Center for Drug Discovery, Northeastern University, Boston, MA02115
| | - Michael N. Teng
- Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL33612
| | - Sami F. Noujaim
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL33612
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13
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Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity. Int J Mol Sci 2022; 23:ijms232314740. [PMID: 36499069 PMCID: PMC9737693 DOI: 10.3390/ijms232314740] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 11/22/2022] [Accepted: 11/23/2022] [Indexed: 11/29/2022] Open
Abstract
Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.
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14
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Matharoo K, Chua J, Park JR, Ingavale S, Jelacic TM, Jurkouich KM, Compton JR, Meinig JM, Chabot D, Friedlander AM, Legler PM. Engineering an Fc-Fusion of a Capsule Degrading Enzyme for the Treatment of Anthrax. ACS Infect Dis 2022; 8:2133-2148. [PMID: 36102590 DOI: 10.1021/acsinfecdis.2c00227] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of B. anthracis. PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade the innate immune responses. CapD is an enzyme that catalyzes the covalent anchoring of PDGA. CapD is an Ntn-amido hydrolase that utilizes an internal Thr-352 as its nucleophile and general acid and base. An internal cleavage produces a free N-terminal Thr-352 and a short and long polypeptide chain. The chains were circularly permuted (CP) to move Thr-352 to the N-terminus of the polypeptide. We previously showed that a branched PEG-CapDS334C-CP could protect mice (80% survival) against a 5 LD50 challenge with B. anthracis Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the in vivo circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapDS334C-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapDS334C-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapDS334C-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the kcat (∼ 2-fold lower). In vivo, the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapDS334C-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapDS334C-CP. The pegylated Fc-fusion was less active than the PEG-CapDS334C-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of B. anthracis CapD (mPEG-Fc-CapD-CP) with activity in vitro, in serum, and on encapsulated bacteria.
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Affiliation(s)
- Khushie Matharoo
- Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22904, United States
| | - Jennifer Chua
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Junyoung R Park
- West Springfield High School, Springfield, Virginia 22152, United States
| | - Susham Ingavale
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Tanya M Jelacic
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Kayla M Jurkouich
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Jaimee R Compton
- Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratories, Washington, D.C. 20375, United States
| | - J Matthew Meinig
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Donald Chabot
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Arthur M Friedlander
- United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, United States
| | - Patricia M Legler
- Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratories, Washington, D.C. 20375, United States
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15
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Jendryczko K, Rzeszotko J, Krzyscik MA, Kocyła A, Szymczyk J, Otlewski J, Szlachcic A. Drug Conjugation via Maleimide-Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies. Mol Pharm 2022; 19:1422-1433. [PMID: 35389227 PMCID: PMC9066409 DOI: 10.1021/acs.molpharmaceut.1c00946] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2021] [Revised: 03/24/2022] [Accepted: 03/25/2022] [Indexed: 11/29/2022]
Abstract
With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody-peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells' proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide-thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol-maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody-drug conjugates.
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Affiliation(s)
- Karolina Jendryczko
- Department
of Protein Engineering, University of Wroclaw, Wroclaw 50-383, Poland
| | - Jakub Rzeszotko
- Department
of Protein Engineering, University of Wroclaw, Wroclaw 50-383, Poland
| | | | - Anna Kocyła
- Department
of Chemical Biology, University of Wroclaw, Wroclaw 50-383, Poland
| | - Jakub Szymczyk
- Department
of Protein Engineering, University of Wroclaw, Wroclaw 50-383, Poland
| | - Jacek Otlewski
- Department
of Protein Engineering, University of Wroclaw, Wroclaw 50-383, Poland
| | - Anna Szlachcic
- Department
of Protein Engineering, University of Wroclaw, Wroclaw 50-383, Poland
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16
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Chen CH, Bepler T, Pepper K, Fu D, Lu TK. Synthetic molecular evolution of antimicrobial peptides. Curr Opin Biotechnol 2022; 75:102718. [PMID: 35395425 DOI: 10.1016/j.copbio.2022.102718] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 02/14/2022] [Accepted: 03/01/2022] [Indexed: 01/18/2023]
Abstract
As we learn more about how peptide structure and activity are related, we anticipate that antimicrobial peptides will be engineered to have strong potency and distinct functions and that synthetic peptides will have new biomedical applications, such as treatments for emerging infectious diseases. As a result of the enormous number of possible amino acid sequences and the low-throughput nature of antimicrobial peptide assays, computational tools for peptide design and optimization are needed for direct experimentation toward obtaining functional sequences. Recent developments in computational tools have improved peptide design, saving labor, reagents, costs, and time. At the same time, improvements in peptide synthesis and experimental platforms continue to reduce the cost and increase the throughput of peptide-drug screening. In this review, we discuss the current methods of peptide design and engineering, including in silico methods and peptide synthesis and screening, and highlight areas of potential improvement.
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Affiliation(s)
- Charles H Chen
- Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
| | - Tristan Bepler
- Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Simons Machine Learning Center, New York Structural Biology Center, New York, NY 10027, USA
| | - Karen Pepper
- Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA
| | - Debbie Fu
- Department of Biology, Tufts University, Medford, MA 02155, USA
| | - Timothy K Lu
- Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), Cambridge, MA 02142, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA 02142, USA; Senti Biosciences, South San Francisco, CA 94080, USA.
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17
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Cavaco M, Castanho MARB, Neves V. The Use of Antibody-Antibiotic Conjugates to Fight Bacterial Infections. Front Microbiol 2022; 13:835677. [PMID: 35330773 PMCID: PMC8940529 DOI: 10.3389/fmicb.2022.835677] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Accepted: 02/14/2022] [Indexed: 12/26/2022] Open
Abstract
The emergence of antimicrobial resistance (AMR) is rapidly increasing and it is one of the significant twenty-first century's healthcare challenges. Unfortunately, the development of effective antimicrobial agents is a much slower and complex process compared to the spread of AMR. Consequently, the current options in the treatment of AMR are limited. One of the main alternatives to conventional antibiotics is the use of antibody-antibiotic conjugates (AACs). These innovative bioengineered agents take advantage of the selectivity, favorable pharmacokinetic (PK), and safety of antibodies, allowing the administration of more potent antibiotics with less off-target effects. Although AACs' development is challenging due to the complexity of the three components, namely, the antibody, the antibiotic, and the linker, some successful examples are currently under clinical studies.
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Affiliation(s)
| | | | - Vera Neves
- Faculdade de Medicina, Instituto de Medicina Molecular João Lobo Antunes, Universidade de Lisboa, Lisbon, Portugal
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18
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Ishikawa S, Ishikawa H, Sato A. Improved Refolding of a Human IgG1 Fc (CH2-CH3) Scaffold from Its Inclusion Body in E. coli by Alkaline Solubilization. Biol Pharm Bull 2022; 45:284-291. [PMID: 35228394 DOI: 10.1248/bpb.b21-00796] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Recently, we developed a platform strategy for hinge-deficient human immunoglobulin G1 (IgG1) Fc fusion as a non-immunostimulatory Fc fusion system. As a starting point to establish a promising approach for generating hinge-deficient Fc fusion proteins in Escherichia (E.) coli, we selected a CH2-CH3 scaffold as a model protein for evaluation. Recombinant CH2-CH3, expressed as inclusion bodies, was solubilized with various denaturants (urea, sarkosyl, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Triton X-100) in neutral (phosphate-buffered saline (PBS), pH 8) or alkaline (50 or 500 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in Chinese hamster ovary (CHO) cells, all denaturants, except urea in CAPS buffer but not in PBS, were found to elicit the dimer formation of solubilized CH2-CH3 on SDS-polyacrylamide gel electrophoresis (PAGE). After dialysis with PBS, sarkosyl-soluble CH2-CH3 inclusion bodies were successfully purified using protein G-Sepharose, indicating their successful refolding. Compared to the purified CH2-CH3 from its sarkosyl-soluble inclusion bodies in neutral buffer, that in 500 mM CAPS alkaline buffer revealed substantial structure-related similarities, such as secondary structures and thermal stabilities, as measured by circular dichroism spectroscopy, to authentic CH2-CH3. Native PAGE analysis also supported the above data. Therefore, solubilization at alkaline pH is an essential factor that promotes the refolding of CH2-CH3. Dimer formation of CH2-CH3 on SDS-PAGE may act as a surrogate marker for its protein refolding status. Our observations may provide important hints toward downstream processing of Fc-fusion production in E. coli.
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Affiliation(s)
- Show Ishikawa
- School of Bioscience and Biotechnology, Tokyo University of Technology
| | - Haruna Ishikawa
- School of Bioscience and Biotechnology, Tokyo University of Technology
| | - Atsushi Sato
- School of Bioscience and Biotechnology, Tokyo University of Technology
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19
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Rashid MH. Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation. MAbs 2022; 14:2111748. [PMID: 36018829 PMCID: PMC9423848 DOI: 10.1080/19420862.2022.2111748] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Although several antibody fragments and antibody fragment-fusion proteins produced in Escherichia coli (E. coli) are approved as therapeutics for various human diseases, a full-length monoclonal or a bispecific antibody produced in E. coli has not yet been approved. The past decade witnessed substantial progress in expression of full-length antibodies in the E. coli cytoplasm and periplasm, as well as in cell-free expression systems. The equivalency of E. coli-produced aglycosylated antibodies and their mammalian cell-produced counterparts, with respect to biochemical and biophysical properties, including antigen binding, in vitro and in vivo serum stability, pharmacokinetics, and in vivo serum half-life, has been demonstrated. Extensive engineering of the Fc domain of aglycosylated antibodies enables recruitment of various effector functions, despite the lack of N-linked glycans. This review summarizes recent research, preclinical advancements, and clinical development of E. coli-produced aglycosylated therapeutic antibodies as monoclonal, bispecific, and antibody-drug conjugates for use in autoimmune, oncology, and immuno-oncology areas.Abbreviations: ADA Anti-drug antibody; ADCC Antibody-dependent cellular cytotoxicity; ADCP Antibody-dependent cellular phagocytosis; ADC Antibody-drug conjugate; aFc Aglycosylated Fc; AMD Age-related macular degeneration aTTP Acquired thrombotic thrombocytopenic purpura; BCMA B-cell maturation antigen; BLA Biologics license application; BsAb Bispecific antibody; C1q Complement protein C1q; CDC Complement-dependent cytotoxicity; CDCC Complement-dependent cellular cytotoxicity; CDCP Complement-dependent cellular phagocytosis; CEX Cation exchange chromatography; CFPS Cell-free protein expression; CHO Chinese Hamster Ovary; CH1-3 Constant heavy chain 1-3; CL Constant light chain; DLBCL Diffuse large B-cell lymphoma; DAR Drug antibody ratio; DC Dendritic cell; dsFv Disulfide-stabilized Fv; EU European Union; EGFR Epidermal growth factor receptor; E. coli Escherichia coli; EpCAM Epithelial cell adhesion molecule; Fab Fragment antigen binding; FACS Fluorescence activated cell sorting; Fc Fragment crystallizable; FcRn Neonatal Fc receptor; FcɣRs Fc gamma receptors; FDA Food and Drug Administration; FL-IgG Full-length immunoglobulin; Fv Fragment variable; FolRαa Folate receptor alpha; gFc Glycosylated Fc; GM-CSF Granulocyte macrophage-colony stimulating factor; GPx7 Human peroxidase 7; HCL Hairy cell leukemia; HIV Human immunodeficiency virusl; HER2 Human epidermal growth factor receptor 2; HGF Hepatocyte growth factor; HIC Hydrophobic interaction chromatography; HLA Human leukocyte antigen; IBs Inclusion bodies; IgG1-4 Immunoglobulin 1-4; IP Intraperitoneal; ITC Isothermal titration calorimetry; ITP Immune thrombocytopenia; IV Intravenous; kDa Kilodalton; KiH Knob-into-Hole; mAb Monoclonal antibody; MAC Membrane-attack complex; mCRC Metastatic colorectal cancer; MM Multipl myeloma; MOA Mechanism of action; MS Mass spectrometry; MUC1 Mucin 1; MG Myasthenia gravis; NB Nanobody; NK Natural killer; nsAA Nonstandard amino acid; NSCLC Non-small cell lung cancer; P. aeruginosa Pseudomonas aeruginosa; PD-1 Programmed cell death 1; PD-L1 Programmed cell death-ligand 1; PDI Protein disulfide isomerase; PECS Periplasmic expression cytometric screening; PK Pharmacokinetics; P. pastoris Pichia pastoris; PTM Post-translational modification; Rg Radius of gyration; RA Rheumatoid arthritis; RT-PCR Reverse transcription polymerase chain reaction; SAXS Small angle X-ray scattering; scF Single chain Fv; SCLC Small cell lung cancer; SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC Size exclusion chromatography; SEED Strand-exchange engineered domain; sRNA Small regulatory RNA; SRP Signal recognition particle; T1/2 Half-life; Tagg Aggregation temperature; TCR T cell receptor; TDB T cell-dependent bispecific; TF Tissue factor; TIR Translation initiation region; Tm Melting temperature; TNBC Triple-negative breast cancer; TNF Tumor necrosis factor; TPO Thrombopoietin; VEGF Vascular endothelial growth factor; vH Variable heavy chain; vL Variable light chain; vWF von Willebrand factor; WT Wild type.
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20
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Jendryczko K, Rzeszotko J, Krzyscik MA, Szymczyk J, Otlewski J, Szlachcic A. Peptibody Based on FGFR1-Binding Peptides From the FGF4 Sequence as a Cancer-Targeting Agent. Front Pharmacol 2021; 12:748936. [PMID: 34867353 PMCID: PMC8636100 DOI: 10.3389/fphar.2021.748936] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Accepted: 09/28/2021] [Indexed: 12/04/2022] Open
Abstract
Targeted therapies are a promising alternative to conventional chemotherapy, with an increasing number of therapeutics targeting specific molecular aberrancies in cancer cells. One of the emerging targets for directed cancer treatments is fibroblast growth factor receptors (FGFRs), which are known to be involved in the pathogenesis and progression of multiple cancer types, specially in lung, bladder, and breast cancers. Here, we are demonstrating the development of the FGFR1-targeting agent based on the interactome screening approach, based on the isolation of binding regions from ligands interacting with the receptor. The parallel analysis by FGFR1 pull-down of chymotryptic peptides coupled with MS analysis, and PepSpot analysis yielded equivalent peptide sequences from FGF4, one of the FGFR1 ligands. Three sequences served as a basis for peptibody (Fc-fusion) generation, to overcome clinical limitations of peptidic agents, and two of them showed favorable FGFR1-binding in vitro and FGFR1-dependent internalization into cells. To validate if developed FGFR1-targeting peptibodies can be used for drug delivery, similar to the well-established concept of antibody-drug conjugates (ADCs), peptibodyF4_1 was successfully conjugated with monomethylauristatin E (MMAE), and has shown significant and specific toxicity toward FGFR1-expressing lung cancer cell lines, with nanomolar EC50 values. Essentially, the development of new effective FGFR1 binders that comprise the naturally occurring FGFR-recognition peptides and Fc region ensuring high plasma stability, and long bloodstream circulation is an interesting strategy expanding targeted anticancer agents' portfolio. Furthermore, identifying peptides effectively binding the receptor from sequences of its ligands is not limited to FGFRs and is an approach versatile enough to be a basis for a new peptide/peptibodies development strategy.
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Affiliation(s)
| | | | | | | | | | - Anna Szlachcic
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland
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21
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Sokolov AV, Dostdar SA, Attwood MM, Krasilnikova AA, Ilina AA, Nabieva AS, Lisitsyna AA, Chubarev VN, Tarasov VV, Schiöth HB. Brain Cancer Drug Discovery: Clinical Trials, Drug Classes, Targets, and Combinatorial Therapies. Pharmacol Rev 2021; 73:1-32. [PMID: 34663683 DOI: 10.1124/pharmrev.121.000317] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Brain cancer is a formidable challenge for drug development, and drugs derived from many cutting-edge technologies are being tested in clinical trials. We manually characterized 981 clinical trials on brain tumors that were registered in ClinicalTrials.gov from 2010 to 2020. We identified 582 unique therapeutic entities targeting 581 unique drug targets and 557 unique treatment combinations involving drugs. We performed the classification of both the drugs and drug targets based on pharmacological and structural classifications. Our analysis demonstrates a large diversity of agents and targets. Currently, we identified 32 different pharmacological directions for therapies that are based on 42 structural classes of agents. Our analysis shows that kinase inhibitors, chemotherapeutic agents, and cancer vaccines are the three most common classes of agents identified in trials. Agents in clinical trials demonstrated uneven distribution in combination approaches; chemotherapy agents, proteasome inhibitors, and immune modulators frequently appeared in combinations, whereas kinase inhibitors, modified immune effector cells did not as was shown by combination networks and descriptive statistics. This analysis provides an extensive overview of the drug discovery field in brain cancer, shifts that have been happening in recent years, and challenges that are likely to come. SIGNIFICANCE STATEMENT: This review provides comprehensive quantitative analysis and discussion of the brain cancer drug discovery field, including classification of drug, targets, and therapies.
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Affiliation(s)
- Aleksandr V Sokolov
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Samira A Dostdar
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Misty M Attwood
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Aleksandra A Krasilnikova
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Anastasia A Ilina
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Amina Sh Nabieva
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Anna A Lisitsyna
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Vladimir N Chubarev
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Vadim V Tarasov
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Helgi B Schiöth
- Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden (A.V.S., S.A.D., M.M.A., H.B.S.); and Department of Pharmacology, Institute of Pharmacy (A.V.S., S.A.D., A.A.K., A.A.I., A.S.N., A.A.L., V.N.C., V.V.T.) and Institute of Translational Medicine and Biotechnology (V.V.T., H.B.S.), I. M. Sechenov First Moscow State Medical University, Moscow, Russia
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22
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Saw PE, Xu X, Kim S, Jon S. Biomedical Applications of a Novel Class of High-Affinity Peptides. Acc Chem Res 2021; 54:3576-3592. [PMID: 34406761 DOI: 10.1021/acs.accounts.1c00239] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Most therapeutic peptides available on the market today are naturally occurring hormones or protein fragments that were serendipitously discovered to possess therapeutic effects. However, the limited repertoire of available natural resources presents difficulties for the development of new peptide drug candidates. Traditional peptides possess several shortcomings that must be addressed for biomedical applications, including relatively low affinity or specificity toward biological targets compared to antibody- and protein scaffold-based affinity molecules, poor in vivo stability owing to rapid enzymatic degradation, and rapid clearance from circulation owing to their small size. Going forward, it will be increasingly important for scientists to develop novel classes of high-affinity and -specificity peptides against desired targets that mitigate these limitations while remaining compatible with pharmaceutical manufacturing processes. Recently, several highly constrained, artificial cyclic peptides have emerged as platforms capable of generating high-affinity peptide binders against various disease-associated protein targets by combining with phage or mRNA display method, some of which have entered clinical trials. In contrast, although linear peptides are relatively easy to synthesize cost-effectively and modify site-specifically at either N- or C-termini compared to cyclic peptides, there have been few linear peptide-based platforms that can provide high-affinity and -specificity peptide binders.In this Account, we describe the creation and development of a novel class of high-affinity peptides, termed "aptide"-from the Latin word "aptus" meaning "to fit" and "peptide"-and summarize their biomedical applications. In the first part, we consider the design and creation of aptides, with a focus on their unique structural features and binding mode, and address screening and identification of target protein-specific aptides. We also discuss advantages of the aptide platform over ordinary linear peptides lacking preorganized structures in terms of the affinity and specificity of identified peptide binders against target molecules. In the second part, we describe the potential biomedical applications of various target-specific aptides, ranging from imaging and therapy to theranostics, according to the types of aptides and diseases. We show that certain aptides can not only bind to a target protein but also inhibit its biological function, thereby showing potential as therapeutics per se. Further, aptides specific for cancer-associated protein antigens can be used as escort molecules or targeting ligands for delivery of chemotherapeutics, cytokine proteins, and nanomedicines, such as liposomes and magnetic particles, to tumors, thereby substantially improving therapeutic effects. Finally, we present a strategy capable of overcoming the critical issue of short blood circulation time associated with most peptides by constructing a hybrid system between an aptide and a hapten cotinine-specific antibody.
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Affiliation(s)
- Phei Er Saw
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, P.R. China
- Biomedical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, P.R. China
| | - Xiaoding Xu
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, P.R. China
- Biomedical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510120, P.R. China
| | - Sunghyun Kim
- Center for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering and Technology (KICET), Cheongju-si 28160, Republic of Korea
| | - Sangyong Jon
- Center for Precision Bio-Nanomedicine, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon 34141, South Korea
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon 34141, South Korea
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23
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Chen F, McDonald V, Newland A. Experts' review: the emerging roles of romiplostim in immune thrombocytopenia (ITP). Expert Opin Biol Ther 2021; 21:1383-1393. [PMID: 34313512 DOI: 10.1080/14712598.2021.1960979] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
INTRODUCTION The management of ITP has in recent years been transformed from reliance on immunosuppressants and splenectomy to targeted therapy with thrombopoietin receptor agonists (TPO-RA) that directly stimulate platelet production in the bone marrow. This has reduced the long-term infective complications and toxicities associated with the use of potent immunosuppressants and splenectomy. The welltolerated romiplostim, itself a novel drug construct called peptibody, has established itself, alongside other TPO-RA as the preferred 2nd line therapy in major international guidelines on treatment of ITP. AREAS COVERED This review summarizes the data from early licensing trials of romiplostim and discusses the real-world experience to date, the unexpected emerging data on treatment-free long-term remission achieved using TPO-RA, and the case for its early introduction in the therapeutic pathway. The emerging risk of thrombosis is also discussed. EXPERT OPINION The use of romiplostim and other TPO-RA will be increasingly brought forward in the management pathway of ITP with the prospect of modifying the long-term outcome of the disease by increasing sustained treatment-free remission. With the prospect of several new targeted therapies been introduced into clinical practice, TPO-RA will likely be a key component of future combination therapies for difficult cases.
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Affiliation(s)
- Frederick Chen
- Department of Clinical Haematology, the Royal London Hospital, Barts Health NHS Trust, London, UK.,Academic Haematology Unit, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and The London School of Medicine and Dentistry, QMUL, London, UK
| | - Vickie McDonald
- Department of Clinical Haematology, the Royal London Hospital, Barts Health NHS Trust, London, UK.,Academic Haematology Unit, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK.,Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry,QMUL, London, UK
| | - Adrian Newland
- Department of Clinical Haematology, the Royal London Hospital, Barts Health NHS Trust, London, UK.,Academic Haematology Unit, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK
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24
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Ning L, Huang J, He B, Kang J. An In Silico Immunogenicity Analysis for PbHRH: An Antiangiogenic Peptibody by Fusing HRH Peptide and Human IgG1 Fc Fragment. Curr Bioinform 2020. [DOI: 10.2174/1574893614666190730104348] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Background:
Peptibodies, the hybrid of peptides and antibodies, represent a novel
strategy in therapeutic use. Previously, we computationally designed an antiangiogenic peptibody
PbHRH, which fused the HRH peptide with angiogenesis-suppressing effect and human IgG1 Fc
fragment using Romiplostim as template. Molecular modeling and simulation results indicated that
it would be a potential drug for the treatment of those angiogenesis related pathological disorders.
However, its immunogenicity is not known.
Methods:
Several bioinformatics tools are used to predict the potential epitopes for the evaluation
of the immunogenicity of PbHRH. Romiplostim is set as the control. IEDB-recommended method
is used in MHC-I and MHC-II binding prediction, and the IEDB web server
(http://tools.iedb.org/immunogenicity/) is used to determine the MHC-I immunogenicity of each
peptide.
Results:
In this work, some peptides are predicted to have the potential ability to bind to MHC-I
and MHC-II molecules both in PbHRH and Romiplostim as the potential epitopes. Most of these
selected peptides are exactly the same. Allele frequency analysis shows a low population
distribution. Combined with the analysis of MHC-I immunogenicity prediction, both HRH and
PbHRH show low immunogenicity.
Conclusion:
Some potential epitopes which could bind to both MHC-I and MHC-II molecules
are predicted using bioinformatics tools. The comparative analysis with Romiplostim and the
results of MHC-I immunogenicity prediction indicate the low immunogenicity of both HRH and
PbHRH. Thus, we form a strategy to evaluate the immunogenicity of peptibodies for the future
improvement.
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Affiliation(s)
- Lin Ning
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Jiang Huang
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Bifang He
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Juanjuan Kang
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
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25
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Jendryczko K, Chudzian J, Skinder N, Opaliński Ł, Rzeszótko J, Wiedlocha A, Otlewski J, Szlachcic A. FGF2-Derived PeptibodyF2-MMAE Conjugate for Targeted Delivery of Cytotoxic Drugs into Cancer Cells Overexpressing FGFR1. Cancers (Basel) 2020; 12:E2992. [PMID: 33076489 PMCID: PMC7602595 DOI: 10.3390/cancers12102992] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2020] [Accepted: 10/10/2020] [Indexed: 12/23/2022] Open
Abstract
Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody-drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody-drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.
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Affiliation(s)
- Karolina Jendryczko
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Julia Chudzian
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Natalia Skinder
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Łukasz Opaliński
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Jakub Rzeszótko
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Antoni Wiedlocha
- Department of Molecular Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, 0379 Oslo, Norway;
- Department of Radiobiology and Radiation Protection, Military Institute of Hygiene and Epidemiology, 01163 Warsaw, Poland
- Center for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway
| | - Jacek Otlewski
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
| | - Anna Szlachcic
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50383 Wroclaw, Poland; (K.J.); (J.C.); (N.S.); (Ł.O.); (J.R.); (J.O.)
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26
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Yetkin-Arik B, Kastelein AW, Klaassen I, Jansen CHJR, Latul YP, Vittori M, Biri A, Kahraman K, Griffioen AW, Amant F, Lok CAR, Schlingemann RO, van Noorden CJF. Angiogenesis in gynecological cancers and the options for anti-angiogenesis therapy. Biochim Biophys Acta Rev Cancer 2020; 1875:188446. [PMID: 33058997 DOI: 10.1016/j.bbcan.2020.188446] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Revised: 10/02/2020] [Accepted: 10/04/2020] [Indexed: 02/06/2023]
Abstract
Angiogenesis is required in cancer, including gynecological cancers, for the growth of primary tumors and secondary metastases. Development of anti-angiogenesis therapy in gynecological cancers and improvement of its efficacy have been a major focus of fundamental and clinical research. However, survival benefits of current anti-angiogenic agents, such as bevacizumab, in patients with gynecological cancer, are modest. Therefore, a better understanding of angiogenesis and the tumor microenvironment in gynecological cancers is urgently needed to develop more effective anti-angiogenic therapies, either or not in combination with other therapeutic approaches. We describe the molecular aspects of (tumor) blood vessel formation and the tumor microenvironment and provide an extensive clinical overview of current anti-angiogenic therapies for gynecological cancers. We discuss the different phenotypes of angiogenic endothelial cells as potential therapeutic targets, strategies aimed at intervention in their metabolism, and approaches targeting their (inflammatory) tumor microenvironment.
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Affiliation(s)
- Bahar Yetkin-Arik
- Ocular Angiogenesis Group, Department of Ophthalmology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands; Department of Medical Biology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands
| | - Arnoud W Kastelein
- Department of Obstetrics and Gynaecology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands.
| | - Ingeborg Klaassen
- Ocular Angiogenesis Group, Department of Ophthalmology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands; Department of Medical Biology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands
| | - Charlotte H J R Jansen
- Department of Obstetrics and Gynaecology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands
| | - Yani P Latul
- Department of Obstetrics and Gynaecology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands
| | - Miloš Vittori
- Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
| | - Aydan Biri
- Department of Obstetrics and Gynecology, Koru Ankara Hospital, Ankara, Turkey
| | - Korhan Kahraman
- Department of Obstetrics and Gynecology, Bahcesehir University School of Medicine, Istanbul, Turkey
| | - Arjan W Griffioen
- Angiogenesis Laboratory, Department of Medical Oncology, Amsterdam UMC, Cancer Center Amsterdam, Amsterdam, the Netherlands
| | - Frederic Amant
- Department of Oncology, KU Leuven, Leuven, Belgium; Center for Gynaecological Oncology, Antoni van Leeuwenhoek, Amsterdam, the Netherlands; Center for Gynaecological Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands; Center for Gynaecological Oncology, Amsterdam University Medical Centers, Amsterdam, the Netherlands
| | - Christianne A R Lok
- Center for Gynaecological Oncology, Antoni van Leeuwenhoek, Amsterdam, the Netherlands
| | - Reinier O Schlingemann
- Ocular Angiogenesis Group, Department of Ophthalmology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands; Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland
| | - Cornelis J F van Noorden
- Department of Medical Biology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands; Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia
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27
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Talotta R, Atzeni F, Laska MJ. Therapeutic peptides for the treatment of systemic lupus erythematosus: a place in therapy. Expert Opin Investig Drugs 2020; 29:845-867. [PMID: 32500750 DOI: 10.1080/13543784.2020.1777983] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
INTRODUCTION Studies in vitro and in vivo have identified several peptides that are potentially useful in treating systemic lupus erythematosus (SLE). The rationale for their use lies in the cost-effective production, high potency, target selectivity, low toxicity, and a peculiar mechanism of action that is mainly based on the induction of immune tolerance. Three therapeutic peptides have entered clinical development, but they have yielded disappointing results. However, some subsets of patients, such as those with the positivity of anti-dsDNA antibodies, appear more likely to respond to these medications. AREAS COVERED This review evaluates the potential use of therapeutic peptides for SLE and gives an opinion on how they may offer advantages for SLE treatment. EXPERT OPINION Given their acceptable safety profile, therapeutic peptides could be added to agents traditionally used to treat SLE and this may offer a synergistic and drug-sparing effect, especially in selected patient populations. Moreover, they could temporarily be utilized to manage SLE flares, or be administered as a vaccine in subjects at risk. Efforts to ameliorate bioavailability, increase the half-life and prevent immunogenicity are ongoing. The formulation of hybrid compounds, like peptibodies or peptidomimetic small molecules, is expected to yield renewed treatments with a better pharmacologic profile and increased efficacy.
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Affiliation(s)
- Rossella Talotta
- Department of Clinical and Experimental Medicine, Rheumatology Unit, Azienda Ospedaliera "Gaetano Martino", University of Messina , Messina, Italy
| | - Fabiola Atzeni
- Department of Clinical and Experimental Medicine, Rheumatology Unit, Azienda Ospedaliera "Gaetano Martino", University of Messina , Messina, Italy
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28
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Rommel MGE, Hoerster K, Milde C, Schenk F, Roser L, Kohlscheen S, Heinz N, Modlich U. Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim. Exp Hematol 2020; 85:33-46.e6. [PMID: 32417303 DOI: 10.1016/j.exphem.2020.04.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2019] [Revised: 04/24/2020] [Accepted: 04/29/2020] [Indexed: 01/01/2023]
Abstract
Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro.
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Affiliation(s)
- Marcel G E Rommel
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Keven Hoerster
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany; Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Christian Milde
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Franziska Schenk
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Luise Roser
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Saskia Kohlscheen
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Niels Heinz
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Ute Modlich
- Research Group for Gene Modification in Stem Cells, Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
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29
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Wang X, Phan MM, Li J, Gill H, Williams S, Gupta N, Quarmby V, Yang J. Molecular Interaction Characterization Strategies for the Development of New Biotherapeutic Antibody Modalities. Antibodies (Basel) 2020; 9:antib9020007. [PMID: 32218192 PMCID: PMC7344756 DOI: 10.3390/antib9020007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 03/12/2020] [Accepted: 03/13/2020] [Indexed: 01/28/2023] Open
Abstract
The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings.
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Affiliation(s)
- Xiangdan Wang
- Department of BioAnalytical Sciences, Genentech, South San Francisco, CA 94080, USA; (M.M.P.); (V.Q.)
- Correspondence: (X.W.); (J.Y.)
| | - Minh Michael Phan
- Department of BioAnalytical Sciences, Genentech, South San Francisco, CA 94080, USA; (M.M.P.); (V.Q.)
| | - Ji Li
- Department of Translational Oncology, Genentech, South San Francisco, CA 94080, USA;
| | - Herman Gill
- Department of Biomedical Imaging, Genentech, South San Francisco, CA 94080, USA; (H.G.); (S.W.)
| | - Simon Williams
- Department of Biomedical Imaging, Genentech, South San Francisco, CA 94080, USA; (H.G.); (S.W.)
| | - Nidhi Gupta
- Department of Immunology, Genentech, South San Francisco, CA 94080, USA;
| | - Valerie Quarmby
- Department of BioAnalytical Sciences, Genentech, South San Francisco, CA 94080, USA; (M.M.P.); (V.Q.)
| | - Jihong Yang
- Department of BioAnalytical Sciences, Genentech, South San Francisco, CA 94080, USA; (M.M.P.); (V.Q.)
- Correspondence: (X.W.); (J.Y.)
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30
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Amann T, Schmieder V, Faustrup Kildegaard H, Borth N, Andersen MR. Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms. Biotechnol Bioeng 2019; 116:2778-2796. [PMID: 31237682 DOI: 10.1002/bit.27101] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Revised: 03/27/2019] [Accepted: 06/18/2019] [Indexed: 12/18/2022]
Abstract
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles. In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.
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Affiliation(s)
- Thomas Amann
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Valerie Schmieder
- acib GmbH-Austrian Centre of Industrial Biotechnology, Graz, Austria.,Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria
| | - Helene Faustrup Kildegaard
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Nicole Borth
- Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria
| | - Mikael Rørdam Andersen
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
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Chen W, Yuan H, Cao W, Wang T, Chen W, Yu H, Fu Y, Jiang B, Zhou H, Guo H, Zhao X. Blocking interleukin-6 trans-signaling protects against renal fibrosis by suppressing STAT3 activation. Am J Cancer Res 2019; 9:3980-3991. [PMID: 31281526 PMCID: PMC6592178 DOI: 10.7150/thno.32352] [Citation(s) in RCA: 141] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Accepted: 04/09/2019] [Indexed: 01/04/2023] Open
Abstract
Rationale: Renal fibrosis is the terminal manifestation of chronic and irreversible renal disease. Effective therapies other than dialysis are extremely limited. In this study, we investigated the potential effects of targeting elevated interleukin-6 (IL-6) levels in the treatment of renal fibrosis. Methods: Fc-gp130 was used to specifically block IL-6 trans-signaling. Unilateral ureteral occlusion (UUO) and ischemia reperfusion (IR) mouse models were constructed to investigate the therapeutic effect of Fc-gp130 on renal fibrosis. The role of IL-6 trans-signaling and phosphorylation of signal transducer and activator of transcription (STAT) 3 in regulating fibroblast accumulation and extracellular matrix protein deposition were evaluated in cell experiments and mouse models. Results: The kidneys of mice with UUO were found to have elevated soluble IL-6 receptor (sIL-6R) levels in the progression of fibrosis. Fc-gp130 attenuated renal fibrosis in mice, as evidenced by reductions in tubular atrophy and the production of extracellular matrix protein. Blockade of IL-6 trans-signaling with Fc-gp130 also reduced inflammation levels, immune cell infiltration, and profibrotic cytokines expression in renal tissue, with decreased STAT3 phosphorylation and reduced fibroblast accumulation in the renal tissue. In vitro, Fc-gp130 also reduced the phosphorylation of STAT3 induced by transforming growth factor (TGF)-β1 in fibroblasts. Furthermore, the therapeutic effect of Fc-gp130 was confirmed in a model of acute kidney injury-chronic kidney disease. Conclusion: Overall, IL-6 trans-signaling may contribute to crucial events in the development of renal fibrosis, and the targeting of IL-6 trans-signaling by Fc-gp130 may provide a novel therapeutic strategy for the treatment of renal fibrosis.
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Ning L, He B, Zhou P, Derda R, Huang J. Molecular Design of Peptide-Fc Fusion Drugs. Curr Drug Metab 2019; 20:203-208. [DOI: 10.2174/1389200219666180821095355] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2017] [Revised: 01/18/2018] [Accepted: 05/29/2018] [Indexed: 12/11/2022]
Abstract
Background:Peptide-Fc fusion drugs, also known as peptibodies, are a category of biological therapeutics in which the Fc region of an antibody is genetically fused to a peptide of interest. However, to develop such kind of drugs is laborious and expensive. Rational design is urgently needed.Methods:We summarized the key steps in peptide-Fc fusion technology and stressed the main computational resources, tools, and methods that had been used in the rational design of peptide-Fc fusion drugs. We also raised open questions about the computer-aided molecular design of peptide-Fc.Results:The design of peptibody consists of four steps. First, identify peptide leads from native ligands, biopanning, and computational design or prediction. Second, select the proper Fc region from different classes or subclasses of immunoglobulin. Third, fuse the peptide leads and Fc together properly. At last, evaluate the immunogenicity of the constructs. At each step, there are quite a few useful resources and computational tools.Conclusion:Reviewing the molecular design of peptibody will certainly help make the transition from peptide leads to drugs on the market quicker and cheaper.
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Affiliation(s)
- Lin Ning
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Bifang He
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Peng Zhou
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
| | - Ratmir Derda
- Department of Chemistry, University of Alberta, Alberta, Canada
| | - Jian Huang
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China
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An innate interaction between IL-18 and the propeptide that inactivates its precursor form. Sci Rep 2019; 9:6160. [PMID: 30992532 PMCID: PMC6467916 DOI: 10.1038/s41598-019-42661-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Accepted: 04/01/2019] [Indexed: 01/03/2023] Open
Abstract
Uncontrolled secretion of mature interleukin (IL)-1β and IL-18 is responsible for severe autoinflammatory or autoimmune disorders and various allergic diseases. Here we report an intramolecular interaction between IL-18 and its propeptide, which is proteolytically removed from its precursor proIL-18 during maturation. The intramolecular interaction was recapitulated intermolecularly using recombinant propeptide. These results suggest the possibility of developing a novel class of peptide-based IL-18 inhibitors that could serve as therapeutic agents for IL-18-related inflammatory diseases.
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Lipok M, Szlachcic A, Kindela K, Czyrek A, Otlewski J. Identification of a peptide antagonist of the FGF1-FGFR1 signaling axis by phage display selection. FEBS Open Bio 2019; 9:914-924. [PMID: 30968602 PMCID: PMC6487701 DOI: 10.1002/2211-5463.12618] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Revised: 02/14/2019] [Accepted: 02/17/2019] [Indexed: 12/11/2022] Open
Abstract
Overexpression of fibroblast growth factor receptor 1 (FGFR1) is a common aberration in lung and breast cancers and has necessitated the design of drugs targeting FGFR1‐dependent downstream signaling and FGFR1 ligand binding. To date, the major group of drugs being developed for treatment of FGFR1‐dependent cancers are small‐molecule tyrosine kinase inhibitors; however, the limited specificity of these drugs has led to increasing attempts to design molecules targeting the extracellular domain of FGFR1. Here, we used the phage display technique to select cyclic peptides F8 (ACSLNHTVNC) and G10 (ACSAKTTSAC) as binders of the fibroblast growth factor 1 (FGF1)–FGFR1 interface. ELISA and in vitro cell assays were performed to reveal that cyclic peptide F8 is more effective in preventing the FGF1–FGFR1 interaction, and also decreases FGF1‐induced proliferation of BA/F3 FGFR1c cells by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Therefore, cyclic peptide F8 can act as a FGF1–FGFR1 interaction antagonist, and may be suitable for further development for potential use in therapies against FGFR1‐expressing cancer cells.
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Affiliation(s)
- Magdalena Lipok
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland.,PORT - Polish Center for Technology Development, Wroclaw, Poland
| | - Anna Szlachcic
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland.,PORT - Polish Center for Technology Development, Wroclaw, Poland
| | - Kinga Kindela
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland
| | - Aleksandra Czyrek
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland
| | - Jacek Otlewski
- Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland.,PORT - Polish Center for Technology Development, Wroclaw, Poland
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Jiang W, Ji M. Receptor tyrosine kinases in PI3K signaling: The therapeutic targets in cancer. Semin Cancer Biol 2019; 59:3-22. [PMID: 30943434 DOI: 10.1016/j.semcancer.2019.03.006] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2018] [Revised: 03/09/2019] [Accepted: 03/28/2019] [Indexed: 12/17/2022]
Abstract
The phosphoinositide 3-kinase (PI3K) pathway, one of the most commonly activated signaling pathways in human cancers, plays a crucial role in the regulation of cell proliferation, differentiation, and survival. This pathway is usually activated by receptor tyrosine kinases (RTKs), whose constitutive and aberrant activation is via gain-of-function mutations, chromosomal rearrangement, gene amplification and autocrine. Blockage of PI3K pathway by targeted therapy on RTKs with tyrosine kinases inhibitors (TKIs) and monoclonal antibodies (mAbs) has achieved great progress in past decades; however, there still remain big challenges during their clinical application. In this review, we provide an overview about the most frequently encountered alterations in RTKs and focus on current therapeutic agents developed to counteract their aberrant functions, accompanied with discussions of two major challenges to the RTKs-targeted therapy in cancer - resistance and toxicity.
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Affiliation(s)
- Wei Jiang
- Department of Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, PR China; Key Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, PR China
| | - Meiju Ji
- Key Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, PR China; Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, PR China.
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Engineering of a GLP-1 analogue peptide/anti-PCSK9 antibody fusion for type 2 diabetes treatment. Sci Rep 2018; 8:17545. [PMID: 30510163 PMCID: PMC6277417 DOI: 10.1038/s41598-018-35869-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Accepted: 11/08/2018] [Indexed: 12/13/2022] Open
Abstract
Type 2 diabetes (T2D) is a complex and progressive disease requiring polypharmacy to manage hyperglycaemia and cardiovascular risk factors. However, most patients do not achieve combined treatment goals. To address this therapeutic gap, we have developed MEDI4166, a novel glucagon-like peptide-1 (GLP-1) receptor agonist peptide fused to a proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralising antibody that allows for glycaemic control and low-density lipoprotein cholesterol (LDL-C) lowering in a single molecule. The fusion has been engineered to deliver sustained peptide activity in vivo in combination with reduced potency, to manage GLP-1 driven adverse effects at high dose, and a favourable manufacturability profile. MEDI4166 showed robust and sustained LDL-C lowering in cynomolgus monkeys and exhibited the anticipated GLP-1 effects in T2D mouse models. We believe MEDI4166 is a novel molecule combining long acting agonist peptide and neutralising antibody activities to deliver a unique pharmacology profile for the management of T2D.
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Shen X, Li Q, Wang F, Bao J, Dai M, Zheng H, Lao X. Generation of a novel long-acting thymosin alpha1-Fc fusion protein and its efficacy for the inhibition of breast cancer in vivo. Biomed Pharmacother 2018; 108:610-617. [DOI: 10.1016/j.biopha.2018.09.064] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2018] [Revised: 09/09/2018] [Accepted: 09/11/2018] [Indexed: 12/21/2022] Open
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Huang Y, Wiedmann MM, Suga H. RNA Display Methods for the Discovery of Bioactive Macrocycles. Chem Rev 2018; 119:10360-10391. [PMID: 30395448 DOI: 10.1021/acs.chemrev.8b00430] [Citation(s) in RCA: 176] [Impact Index Per Article: 25.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The past two decades have witnessed the emergence of macrocycles, including macrocyclic peptides, as a promising yet underexploited class of de novo drug candidates. Both rational/computational design and in vitro display systems have contributed tremendously to the development of cyclic peptide binders of either traditional targets such as cell-surface receptors and enzymes or challenging targets such as protein-protein interaction surfaces. mRNA display, a key platform technology for the discovery of cyclic peptide ligands, has become one of the leading strategies that can generate natural-product-like macrocyclic peptide binders with antibody-like affinities. On the basis of the original cell-free transcription/translation system, mRNA display is highly evolvable to realize its full potential by applying genetic reprogramming and chemical/enzymatic modifications. In addition, mRNA display also allows the follow-up hit-to-lead development using high-throughput focused affinity maturation. Finally, mRNA-displayed peptides can be readily engineered to create chemical conjugates based on known small molecules or biologics. This review covers the birth and growth of mRNA display and discusses the above features of mRNA display with success stories and future perspectives and is up to date as of August 2018.
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Affiliation(s)
- Yichao Huang
- Department of Chemistry, Graduate School of Science , The University of Tokyo , 7-3-1 Hongo , Bunkyo-ku, Tokyo 113-0033 , Japan
| | - Mareike Margarete Wiedmann
- Department of Chemistry, Graduate School of Science , The University of Tokyo , 7-3-1 Hongo , Bunkyo-ku, Tokyo 113-0033 , Japan
| | - Hiroaki Suga
- Department of Chemistry, Graduate School of Science , The University of Tokyo , 7-3-1 Hongo , Bunkyo-ku, Tokyo 113-0033 , Japan
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39
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40
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Ning L, Li Z, Bai Z, Hou S, He B, Huang J, Zhou P. Computational Design of Antiangiogenic Peptibody by Fusing Human IgG1 Fc Fragment and HRH Peptide: Structural Modeling, Energetic Analysis, and Dynamics Simulation of Its Binding Potency to VEGF Receptor. Int J Biol Sci 2018; 14:930-937. [PMID: 29989101 PMCID: PMC6036755 DOI: 10.7150/ijbs.24582] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2017] [Accepted: 01/24/2018] [Indexed: 11/05/2022] Open
Abstract
Peptibodies represent a new class of biological therapeutics with combination of peptide activity and antibody-like properties. Previously, we discovered a novel peptide HRH that exhibited a dose-dependent angiogenesis-suppressing effect by targeting vascular endothelial growth factor receptors (VEGFRs). Here, we computationally designed an antiangiogenic peptibody, termed as PbHRH, by fusing the HRH peptide to human IgG1 Fc fragment using the first approved peptibody drug Romiplostim as template. The biologically active peptide of Romiplostim is similar with HRH peptide; both of them have close sequence lengths and can fold into a α-helical conformation in free state. Molecular dynamics simulations revealed that the HRH functional domain is highly flexible, which is functionally independent of Fc fragment in the designed PbHRH peptibody. Subsequently, the intermolecular interactions between VEGFR-1 domain 2 (D2) and PbHRH were predicted, clustered and refined into three representatives. Conformational analysis and energetic evaluation unraveled that the PbHRH can adopt multiple binding modes to block the native VEGF-A binding site of VEGFR-1 D2 with its HRH functional domain, although the binding effectiveness of HRH segments in peptibody context seems to be moderately decreased relative to that of free HRH peptide. Overall, it is suggested that integrating HRH peptide into PbHRH peptibody does not promote the direct intermolecular interaction between VEGFR-1 D2 and HRH. Instead, the peptibody may indirectly help to improve the pharmacokinetic profile and bioavailability of HRH.
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Affiliation(s)
- Lin Ning
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Zhongyan Li
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Zhengya Bai
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Shasha Hou
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Bifang He
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Jian Huang
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
| | - Peng Zhou
- Center for Informational Biology, University of Electronic Science and Technology of China (UESTC), Chengdu 611731, China
- School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, China
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Zvonova EA, Tyurin AA, Soloviev AA, Goldenkova-Pavlova IV. Strategies for Modulation of Pharmacokinetics of Recombinant Therapeutic Proteins. ACTA ACUST UNITED AC 2018. [DOI: 10.1134/s2079086418020093] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Frutos S, Jordan JB, Bio MM, Muir TW, Thiel OR, Vila-Perelló M. Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation. Org Biomol Chem 2018; 14:9549-9553. [PMID: 27722696 DOI: 10.1039/c6ob01833e] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.
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Affiliation(s)
- S Frutos
- ProteoDesign S.L., Baldiri Reixac 10-12, 08028 Barcelona, Spain.
| | - J B Jordan
- Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
| | - M M Bio
- Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
| | - T W Muir
- Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
| | - O R Thiel
- Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
| | - M Vila-Perelló
- ProteoDesign S.L., Baldiri Reixac 10-12, 08028 Barcelona, Spain.
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44
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Lyophilization: Process Design, Robustness, and Risk Management. CHALLENGES IN PROTEIN PRODUCT DEVELOPMENT 2018. [DOI: 10.1007/978-3-319-90603-4_19] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Cavaco M, Castanho MARB, Neves V. Peptibodies: An elegant solution for a long-standing problem. Biopolymers 2017; 110. [PMID: 29266205 DOI: 10.1002/bip.23095] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2017] [Revised: 11/20/2017] [Accepted: 11/24/2017] [Indexed: 01/09/2023]
Abstract
Chimeric proteins composed of a biologically active peptide and a fragment crystallizable (Fc) domain of immunoglobulin G (IgG) are known as peptibodies. They present an extended half-life due to neonatal Fc receptor (FcRn) salvage pathway, a decreased renal clearance rate owing to its increased size (≈70 kDa) and, depending on the peptide used in the design of the peptibody, an active-targeting moiety. Also, the peptides therapeutic activity is boosted by the number of peptides in the fusion protein (at least two peptides) and to some peptides' alterations. Peptibodies are mainly obtained through recombinant DNA technology. However, to improve peptide properties, "unnatural" changes have been introduced to the original peptides' sequence, for instance, the incorporation of D- or non-natural amino acid residues or even cyclization thus, limiting the application of genetic engineering in the production of peptibodies, since these peptides must be obtained via chemical synthesis. This constrains prompted the development of new methods for conjugation of peptides to Fc domains. Another challenge, subject of intense research, relates to the large-scale production of such peptibodies using these new techniques, which can be minimized by their proved value. To date, two peptibodies, romiplostim and dulaglutide, have been approved and stay as the standard of care in their areas of action. Furthermore, a considerable number of peptibodies are currently in preclinical and clinical development.
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Affiliation(s)
- Marco Cavaco
- Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa, 1649-028, Portugal
| | - Miguel A R B Castanho
- Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa, 1649-028, Portugal
| | - Vera Neves
- Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisboa, 1649-028, Portugal
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Zhu X, Yang J, Gao Y, Wu C, Yi L, Li G, Qi Y. The dual effects of a novel peptibody on angiogenesis inhibition and M2 macrophage polarization on sarcoma. Cancer Lett 2017; 416:1-10. [PMID: 29104145 DOI: 10.1016/j.canlet.2017.10.043] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2017] [Revised: 10/25/2017] [Accepted: 10/27/2017] [Indexed: 10/18/2022]
Abstract
Inhibition of the VEGF/VEGF receptor (VEGFR) and angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway is a potential target for tumor angiogenesis. We previously showed that a peptide AS16 which dually inhibits VEGFR/Ang-2 could reduce the tumor growth and decrease the number of microvessels in tumor. However, its short circulating half-life in the serum limits its clinical applications. In this study, as an effort to prolong the short in vivo half-life of AS16, we designed a fusion protein containing peptide AS16 and an IgG Fc fragment. Pharmacokinetic study also revealed that AS16-Fc has a prolonged circulating half-life of about 231 min in rats. We examined the effects of treatment on the tumor vasculature and immune cell populations, tumor growth, in both the MCA-205 and S180 tumor models. We found that AS16-Fc dramatically reduced tumor volume, vascular density and tumor-associated macrophages. Macrophages were identified as potential novel targets following anti-angiogenic therapy, our findings imply a novel role for anti-angiogenic peptide AS16-Fc. These findings indicate that AS16-Fc could be more effective on inhibiting tumor growth angiogenesis and tumor immune microenvironment than that of peptide AS16.
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Affiliation(s)
- Xiaoqing Zhu
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Jiali Yang
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Yanfeng Gao
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Chunjing Wu
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Lili Yi
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Guodong Li
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China; Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China.
| | - Yuanming Qi
- School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China; Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China.
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Haußner C, Lach J, Eichler J. Synthetic antibody mimics for the inhibition of protein-ligand interactions. Curr Opin Chem Biol 2017; 40:72-77. [PMID: 28735229 DOI: 10.1016/j.cbpa.2017.07.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Revised: 05/31/2017] [Accepted: 07/03/2017] [Indexed: 12/22/2022]
Abstract
The rational/structure-based design and/or combinatorial development of molecules capable of selectively binding to a protein, represents a promising strategy for a range of biomedical applications, in particular the inhibition of disease-associated protein-ligand interactions. The design of such protein binding molecules is often based on an antibody against the target protein, or involves the generation of smaller molecules that retain the binding characteristics of the antibody. Alternatively, protein binding molecules can be selected from protein libraries based on small, stably folded protein scaffolds presenting flexible loops, which are randomized in the libraries. In addition to recombinantly synthesized molecules, synthetic antibody paratope mimetic peptides have emerged as promising molecules for the design of antibody mimics.
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Affiliation(s)
- Christina Haußner
- Department of Chemistry and Pharmacy, University of Erlangen-Nurnberg, Schuhstr. 19, 91052 Erlangen, Germany
| | - Johannes Lach
- Department of Chemistry and Pharmacy, University of Erlangen-Nurnberg, Schuhstr. 19, 91052 Erlangen, Germany
| | - Jutta Eichler
- Department of Chemistry and Pharmacy, University of Erlangen-Nurnberg, Schuhstr. 19, 91052 Erlangen, Germany.
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CLEC14a-HSP70-1A interaction regulates HSP70-1A-induced angiogenesis. Sci Rep 2017; 7:10666. [PMID: 28878328 PMCID: PMC5587741 DOI: 10.1038/s41598-017-11118-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2017] [Accepted: 08/18/2017] [Indexed: 12/31/2022] Open
Abstract
CLEC14a (C-type lectin domain family 14 member) is a tumor endothelial cell marker protein that is known to play an important role in tumor angiogenesis, but the basic molecular mechanisms underlying this function have not yet been clearly elucidated. In this study, using various proteomic tools, we isolated a 70-kDa protein that interacts with the C-type lectin-like domain of CLEC14a (CLEC14a-CTLD) and identified it as heat shock protein 70-1A (HSP70-1A). Co-immunoprecipitation showed that HSP70-1A and CLEC14a interact on endothelial cells. In vitro binding analyses identified that HSP70-1A specifically associates with the region between amino acids 43 and 69 of CLEC14a-CTLD. Competitive blocking experiments indicated that this interacting region of CLEC14a-CTLD significantly inhibits HSP70-1A-induced extracellular signal-regulated kinase (ERK) phosphorylation and endothelial tube formation by directly inhibiting CLEC14a-CTLD-mediated endothelial cell-cell contacts. Our data suggest that the specific interaction of HSP70-1A with CLEC14a may play a critical role in HSP70-1A-induced angiogenesis and that the HSP70-1A-interacting region of CLEC14a-CTLD may be a useful tool for inhibiting HSP70-1A-induced angiogenesis.
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Spahr C, Gunasekaran K, Walker KW, Shi SDH. High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein. MAbs 2017; 9:812-819. [PMID: 28506197 DOI: 10.1080/19420862.2017.1325556] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first 2 amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS2 with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH4, thus eliminating several possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS2 and MS3 using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS3 using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.
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Affiliation(s)
- Chris Spahr
- a Discovery Attribute Sciences, Therapeutic Discovery, Amgen Inc. , Thousand Oaks , CA , USA
| | - Kannan Gunasekaran
- b Biologics Optimization, Therapeutic Discovery, Amgen Inc. , Thousand Oaks , CA , USA
| | - Kenneth W Walker
- b Biologics Optimization, Therapeutic Discovery, Amgen Inc. , Thousand Oaks , CA , USA
| | - Stone D-H Shi
- a Discovery Attribute Sciences, Therapeutic Discovery, Amgen Inc. , Thousand Oaks , CA , USA
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Alizadeh AA, Hamzeh-Mivehroud M, Farajzadeh M, Dastmalchi S. Identification of novel peptides against TNF-α using phage display technique and in silico modeling of their modes of binding. Eur J Pharm Sci 2017; 96:490-498. [DOI: 10.1016/j.ejps.2016.10.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Revised: 10/03/2016] [Accepted: 10/04/2016] [Indexed: 01/15/2023]
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