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Lee CH, Wu CJ, Yen FY, Chiang JY, Shen TJ, Leu SJ, Chang CR, Lo HJ, Tsai BY, Mao YC, Andriani V, Thenaka PC, Wang WC, Chao YP, Yang YY. Identification of chicken-derived antibodies targeting the Candida albicans Als3 protein. Appl Microbiol Biotechnol 2025; 109:85. [PMID: 40198376 PMCID: PMC11978541 DOI: 10.1007/s00253-025-13469-3] [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: 01/23/2025] [Revised: 03/21/2025] [Accepted: 03/24/2025] [Indexed: 04/10/2025]
Abstract
Candida albicans is a major opportunistic pathogen, responsible for nearly half of clinical candidemia cases. The rising prevalence of azole-resistant Candida species represents a significant clinical challenge, underscoring the urgent need for alternative therapeutic strategies. Monoclonal antibody-based therapies have emerged as a promising and cost-effective approach to combating Candida infections. Agglutinin-like sequence protein 3 (Als3), a key cell surface protein of C. albicans, plays a pivotal role in adherence and biofilm formation, both of which are essential for its pathogenesis. In this study, recombinant Als3 protein was purified and utilized to immunize chickens, resulting in the production of Als3-specific immunoglobulin Y (IgY) antibodies. Two single-chain variable fragment (scFv) antibody libraries were subsequently constructed using phage display technology, yielding transformant counts of 5.3 × 107 and 2.8 × 107, respectively. Phage-based enzyme-linked immunosorbent assay (ELISA) revealed enhanced signals following bio-panning, enabling the identification and sequence validation of three scFv antibodies. These scFv antibodies exhibited strong binding activities to Als3, as confirmed through ELISA and western blot analyses. Binding affinities were determined to be ~ 10⁻⁸ M via serial titration ELISA and competitive ELISA. Additionally, the selected scFv antibodies specifically recognized endogenous Als3 protein in C. albicans, as demonstrated by western blot and cell-based ELISA assays. In conclusion, this study successfully generated and characterized high-affinity scFv antibodies targeting Als3, which exhibited exceptional specificity and binding activity. These findings highlight their potential as promising immunotherapeutic candidates for the treatment of C. albicans infections. KEY POINTS: • The Als3 protein of C. albicans is a critical biomarker and therapeutic target • Chicken-derived scFv antibodies against Als3 were developed via phage display • The scFv antibodies showed strong binding to endogenous Als3 in C. albicans.
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Affiliation(s)
- Chi-Hsin Lee
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University Inc., Taipei, 110301, Taiwan
- Core Laboratory of Antibody Generation and Research, Taipei Medical University, Taipei, 110301, Taiwan
| | - Chao-Jung Wu
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University Inc., Taipei, 110301, Taiwan
| | - Fang-Yi Yen
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
| | - Jia-Yun Chiang
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
| | - Ting-Jing Shen
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University Inc., Taipei, 110301, Taiwan
| | - Sy-Jye Leu
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 110301, Taiwan
| | - Chuang-Rung Chang
- Institute of Biotechnology, National Tsing Hua University, Hsinchu, 300044, Taiwan
| | - Hsiu-Jung Lo
- National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, 350401, Taiwan
| | - Bor-Yu Tsai
- Navi Bio-Therapeutics Inc., Taipei, 10351, Taiwan
| | - Yan-Chiao Mao
- Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung, 407219, Taiwan
| | - Valencia Andriani
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
| | - Priskila Cherisca Thenaka
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan
| | - Wei-Chu Wang
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University Inc., Taipei, 110301, Taiwan
| | - Yu-Pin Chao
- iReal Biotechnology Inc., Hsinchu, 30060, Taiwan
| | - Yi-Yuan Yang
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No. 301, Yuantong Rd., Zhonghe Dist., New Taipei City, 235, Taiwan.
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University Inc., Taipei, 110301, Taiwan.
- Core Laboratory of Antibody Generation and Research, Taipei Medical University, Taipei, 110301, Taiwan.
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Soerensen A, Popovic F, Olesen CH, Mendez BL, Lohse B, Chen Z, Farci P, Purcell RH, Alter HJ, Barfod LK, Bukh J, Prentoe J. Selection and characterization of a broadly neutralizing class of HCV anti-E2 VH1-69 antibodies. PLoS Pathog 2025; 21:e1012428. [PMID: 40153382 DOI: 10.1371/journal.ppat.1012428] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Accepted: 02/10/2025] [Indexed: 03/30/2025] Open
Abstract
Identification and characterization of antibody epitope targets on the hepatitis C virus (HCV) envelope proteins remain crucial for developing an effective vaccine. Building on prior research defining E1/E2 antibody epitope clustering, we screened a phage display library derived from a chronic HCV patient against detergent-extracted full-length E1/E2 from both the patient's acute-phase isolate (H77, genotype 1a) and a heterologous isolate (S52, genotype 3a). This approach yielded a panel of VH1-69 derived antibody fragments (Fabs) with similar characteristics. Interestingly, all members of the panel exhibited blocking activity against both antigenic region 2 and 3 (AR2 and AR3) in competition ELISAs, which contrasts with the behavior of most previously identified AR3-targeting antibodies. The VH1-69 derived binders had a high affinity for soluble E2 in both Fab and IgG formats, with dissociation constants in the low picomolar range. These Fab binders were broadly neutralizing against a panel of HCV cell culture viruses of genotype 1-6 with higher potency than the well-characterized reference Fab, AR3A. However, in the IgG format the antibodies had similar potency. These findings expand our understanding of potential targets for vaccine development by characterizing a panel of antibodies targeting an AR3 epitope also involving or occluding the back layer of E2. The broad neutralization and high affinity of these antibodies suggest a benefit to targeting both the back layer and the front layer of E2 in HCV vaccine designs to expand the repertoire of broadly neutralizing antibodies, thereby offering promise for the development of more effective preventive measures against this pervasive human pathogen.
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Affiliation(s)
- Andreas Soerensen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
| | - Filip Popovic
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
| | - Christina Holmboe Olesen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
| | - Blanca Lopez Mendez
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Brian Lohse
- Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Zhaochun Chen
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Patrizia Farci
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Robert H Purcell
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Harvey J Alter
- Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Lea Klingenberg Barfod
- Centre for Translational Medicine and Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
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Babaei Khorzoughi S, Tavakoli M, Mortazavi M, Jafarnejad Z, Malekpour A, Kopaiee Malek T, Kargar F. A review of recombinant HER3 affibodies with an effective diagnostic view of cancer cells. J Drug Target 2025; 33:316-327. [PMID: 39485069 DOI: 10.1080/1061186x.2024.2420202] [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: 07/05/2024] [Revised: 09/10/2024] [Accepted: 10/16/2024] [Indexed: 11/03/2024]
Abstract
Breast cancer is one of the leading causes of cancer-related deaths among women globally. Factors like increased expression of HER family members contribute to its development, with elevated HER3 levels-especially in conjunction with tyrosine kinase receptors like HER2-playing a critical role in activating cancer pathways essential for cell survival and proliferation. Detecting high HER3 levels is vital for effective treatment. Affibody proteins, a class that includes antibodies, are used to identify elevated HER3 expression due to their high binding affinity. These innovative non-immune probes show promise in therapy, diagnostics, and biotechnology because of their exceptional specificity and affinity for target proteins. The design of recombinant affibodies enhances HER3 detection accuracy and supports the development of targeted therapies. Advanced engineering techniques optimize these affibodies for stability and binding efficacy, making them suitable for clinical applications. Additionally, their versatility allows integration with imaging technologies for real-time monitoring of HER3 expression and therapeutic responses. This comprehensive approach could lead to more personalized treatment options for patients with HER3-positive breast cancers, improving patient management and outcomes. This study presents recombinant affibodies designed to bind HER3 for cancer cell identification and introduces novel methods for producing various affibody molecules.
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Affiliation(s)
- Sahar Babaei Khorzoughi
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | - Mehrnoosh Tavakoli
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | - Mojtaba Mortazavi
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | - Zahra Jafarnejad
- Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
| | | | - Tara Kopaiee Malek
- Department of Cell and Molecular Biology, Faculty of Science, Azad University of Damghan, Damghan, Iran
| | - Farzane Kargar
- Department of Medical Biotechnology, School of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
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Czarnecka M, Findik N, Schlör A, Hanack K. Development of an optimized cell-based selection system for phage display libraries. Biol Methods Protoc 2025; 10:bpaf009. [PMID: 39968222 PMCID: PMC11835232 DOI: 10.1093/biomethods/bpaf009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2024] [Revised: 01/08/2025] [Accepted: 02/05/2025] [Indexed: 02/20/2025] Open
Abstract
The discovery of antibodies through phage display is significantly influenced by antigen presentation during panning, particularly for membrane-anchored proteins, which pose challenges due to their complex structures. Traditional approaches, such as whole cells expressing the target protein, often result in low antigen density and high background signals. In this study, we describe an alternative method using stably transfected cell lines that express the target antigen on their surface, regulated by an intracellular enhanced green fluorescent protein (EGFP) signal. This system enables high-throughput flow cytometry-based screening of phage display libraries to isolate human antibodies that recognize the native conformation of membrane proteins. Using human epithelial cell adhesion molecule (EpCAM) and human neuroplastin 65 (NP65) as model antigens, we established an optimized screening workflow with polyclonal phage pools. Selected EpCAM-specific single-chain variable fragments (scFvs) from a naïve library were recombinantly expressed with an IgG4 scaffold and characterized for specific binding. This approach provides an effective platform for the identification of antibodies against membrane proteins in their native state.
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Affiliation(s)
- Malgorzata Czarnecka
- Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam, Germany
| | - Nicole Findik
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
| | - Anja Schlör
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
| | - Katja Hanack
- Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam, Germany
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
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5
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Chen Z, Feng L, Wang L, Zhang L, Zheng B, Fu H, Li F, Liu L, Lv Q, Deng R, Xu Y, Hu Y, Zheng J, Qin C, Bao L, Wang X, Jin Q. A broadly neutralizing antibody against the SARS-CoV-2 Omicron sub-variants BA.1, BA.2, BA.2.12.1, BA.4, and BA.5. Signal Transduct Target Ther 2025; 10:14. [PMID: 39800731 PMCID: PMC11725571 DOI: 10.1038/s41392-024-02114-6] [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: 01/22/2024] [Revised: 11/29/2024] [Accepted: 12/24/2024] [Indexed: 01/16/2025] Open
Abstract
The global spread of Severe Acute Respiratory Syndrome Coronavirus 2. (SARS-CoV-2) and its variant strains, including Alpha, Beta, Gamma, Delta, and now Omicron, pose a significant challenge. With the constant evolution of the virus, Omicron and its subtypes BA.1, BA.2, BA.3, BA.4, and BA.5 have developed the capacity to evade neutralization induced by previous vaccination or infection. This evasion highlights the urgency in discovering new monoclonal antibodies (mAbs) with neutralizing activity, especially broadly neutralizing antibodies (bnAbs), to combat the virus.In the current study, we introduced a fully human neutralizing mAb, CR9, that targets Omicron variants. We demonstrated the mAb's effectiveness in inhibiting Omicron replication both in vitro and in vivo. Structural analysis using cryo-electron microscopy (cryo-EM) revealed that CR9 binds to an epitope formed by RBD residues, providing a molecular understanding of its neutralization mechanism. Given its potency and specificity, CR9 holds promise as a potential adjunct therapy for treating Omicron infections. Our findings highlight the importance of continuous mAb discovery and characterization in addressing the evolving threat of COVID-19.
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MESH Headings
- SARS-CoV-2/immunology
- Humans
- Antibodies, Viral/immunology
- Antibodies, Viral/chemistry
- COVID-19/immunology
- COVID-19/virology
- Animals
- Antibodies, Neutralizing/immunology
- Antibodies, Neutralizing/chemistry
- Antibodies, Neutralizing/genetics
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/chemistry
- Mice
- Spike Glycoprotein, Coronavirus/immunology
- Spike Glycoprotein, Coronavirus/genetics
- Spike Glycoprotein, Coronavirus/chemistry
- Cryoelectron Microscopy
- Chlorocebus aethiops
- Betacoronavirus/immunology
- Betacoronavirus/genetics
- Broadly Neutralizing Antibodies/immunology
- Broadly Neutralizing Antibodies/genetics
- Vero Cells
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Affiliation(s)
- Zhe Chen
- NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China
| | - Leilei Feng
- CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Lei Wang
- CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Li Zhang
- Department of Vaccine Clinical Evaluation, Jiangsu Provincial Center for Disease Prevention and Control, 172 Jiangsu Road, Gulou Qu, Nanjing, 210009, China
| | - Binyang Zheng
- Department of Vaccine Clinical Evaluation, Jiangsu Provincial Center for Disease Prevention and Control, 172 Jiangsu Road, Gulou Qu, Nanjing, 210009, China
| | - Hua Fu
- NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China
| | - Fengdi Li
- Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center, Peking Union Medical Collage (PUMC), Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China
| | - Ligai Liu
- Beijing Ditan Hospital, Capital Medical University, Beijing, 100015, PR China
| | - Qi Lv
- Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center, Peking Union Medical Collage (PUMC), Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China
| | - Ran Deng
- Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center, Peking Union Medical Collage (PUMC), Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China
| | - YanLi Xu
- Chronic Disease Management Center, Beijing Ditan Hospital, Capital Medical University, Beijing, 100015, PR China
| | - Yongfeng Hu
- NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China
| | - Jianhua Zheng
- NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China
| | - Chuan Qin
- Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center, Peking Union Medical Collage (PUMC), Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China
| | - Linlin Bao
- Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center, Peking Union Medical Collage (PUMC), Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China.
| | - Xiangxi Wang
- CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
| | - Qi Jin
- NHC Key Laboratory of Systems Biology of Pathogens, State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, China.
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Hsieh SJ, Tsai TH, Lin JH, Lin TY, Chang FL, Chiang CW, Li PJ, Zheng JH, Tsai KC, Hung CS, Lee YC. Characterization of anti-EBNA-1 antibodies and exploration of their molecular mimicry potential in an EBV-infected Sjögren's syndrome patient. Biochem Biophys Res Commun 2024; 735:150839. [PMID: 39427375 DOI: 10.1016/j.bbrc.2024.150839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 10/12/2024] [Accepted: 10/15/2024] [Indexed: 10/22/2024]
Abstract
There is a potential link between autoimmune diseases and Epstein-Barr virus (EBV) infections, with EBV playing a substantial role in the onset of Sjögren's syndrome (SjS). Some EBV proteins could mimic host self-antigens post-infection, leading to molecular mimicry. This similarity may cause the immune system to attack its tissues mistakenly. Among the various proteins associated with EBV, nuclear antigen 1 (EBNA-1) is essential for the latent replication of infected cells and is prevalent in all EBV-related diseases. In the study, single-chain variable fragment (scFv) antibodies targeting EBNA-1 were isolated using phage display technology from a primary SjS patient who also had a chronic active EBV infection. The specific clones were enriched after panning, and the binding activity of selected scFvs targeting EBNA-1 was confirmed. Sequence analysis indicated that the scFvs exhibiting positive signals could be grouped into five clones, all of which used homologous heavy chain V regions derived from germline Vh4-39, and two types of light chain V regions stemming from germline Vλ1-44 and Vλ3-15. These scFvs were found to exhibit a high degree of somatic mutations, likely indicative of antigen selection. Of the scFvs, P1-3 demonstrated the strongest binding affinity to EBNA-1, exhibiting a determined value of 7.3 x 10-8 M, and showed cross-reactivity to the SjS associated La/SSB self-antigen. The experimental results combined with AlphaFold 3 predictions revealed a potential epitope for scFv P1-3 binding to EBNA-1. Additionally, scFv P1-3 could also cross-bind to the modeled structure of La/SSB. We inferred a possible structural correlation between EBNA-1 and La/SSB involving an X2AX6PG epitope motif. This research contributes to our understanding of the structural basis of the interactions between antibodies and EBNA-1, shedding light on the VH and VL gene usage of anti-EBNA-1 antibodies in EBV-infected SjS patients and the potential origins of autoantibodies.
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Affiliation(s)
- Shang-Ju Hsieh
- Division of Urology, Department of Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan
| | - Tsung-Hsun Tsai
- Department of Psychiatry, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan
| | - Jiun-Han Lin
- Department of Industrial Technology, Ministry of Economic Affairs, Taipei, Taiwan; Food Industry Research and Development Institute, Hsinchu City, Taiwan
| | - Tsai-Yu Lin
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Fu-Ling Chang
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
| | - Chen-Wei Chiang
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Pei Jhen Li
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
| | - Jia Huei Zheng
- Taiwan Autoantibody Biobank Initiative, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan
| | - Keng-Chang Tsai
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan; Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Ching-Sheng Hung
- Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.
| | - Yu-Ching Lee
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan; Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
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7
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Istomina PV, Gorchakov AA, Paoin C, Yamabhai M. Phage display for discovery of anticancer antibodies. N Biotechnol 2024; 83:205-218. [PMID: 39186973 DOI: 10.1016/j.nbt.2024.08.506] [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: 02/24/2024] [Revised: 08/20/2024] [Accepted: 08/20/2024] [Indexed: 08/28/2024]
Abstract
Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development.
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Affiliation(s)
- Polina V Istomina
- Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand
| | - Andrey A Gorchakov
- Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences, Lavrentieva 8/2, Novosibirsk 630090, Russia
| | - Chatchanok Paoin
- Medical Oncology Division, Institute of Medicine, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand
| | - Montarop Yamabhai
- Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand.
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8
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Aboul-Ella H, Gohar A, Ali AA, Ismail LM, Mahmoud AEER, Elkhatib WF, Aboul-Ella H. Monoclonal antibodies: From magic bullet to precision weapon. MOLECULAR BIOMEDICINE 2024; 5:47. [PMID: 39390211 PMCID: PMC11467159 DOI: 10.1186/s43556-024-00210-1] [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: 05/05/2024] [Accepted: 09/19/2024] [Indexed: 10/12/2024] Open
Abstract
Monoclonal antibodies (mAbs) are used to prevent, detect, and treat a broad spectrum of non-communicable and communicable diseases. Over the past few years, the market for mAbs has grown exponentially with an expected compound annual growth rate (CAGR) of 11.07% from 2024 (237.64 billion USD estimated at the end of 2023) to 2033 (679.03 billion USD expected by the end of 2033). Ever since the advent of hybridoma technology introduced in 1975, antibody-based therapeutics were realized using murine antibodies which further progressed into humanized and fully human antibodies, reducing the risk of immunogenicity. Some benefits of using mAbs over conventional drugs include a drastic reduction in the chances of adverse reactions, interactions between drugs, and targeting specific proteins. While antibodies are very efficient, their higher production costs impede the process of commercialization. However, their cost factor has been improved by developing biosimilar antibodies as affordable versions of therapeutic antibodies. Along with the recent advancements and innovations in antibody engineering have helped and will furtherly help to design bio-better antibodies with improved efficacy than the conventional ones. These novel mAb-based therapeutics are set to revolutionize existing drug therapies targeting a wide spectrum of diseases, thereby meeting several unmet medical needs. This review provides comprehensive insights into the current fundamental landscape of mAbs development and applications and the key factors influencing the future projections, advancement, and incorporation of such promising immunotherapeutic candidates as a confrontation approach against a wide list of diseases, with a rationalistic mentioning of any limitations facing this field.
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Affiliation(s)
- Hassan Aboul-Ella
- Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
| | - Asmaa Gohar
- Department of Microbiology and Immunology, Faculty of Pharmacy, Galala University, Suez, Egypt
- Department of Microbiology and Immunology, Faculty of Pharmacy, Ahram Canadian University (ACU), Giza, Egypt
- Egyptian Drug Authority (EDA), Giza, Egypt
| | - Aya Ahmed Ali
- Department of Microbiology and Immunology, Faculty of Pharmacy, Sinai University, Sinai, Egypt
| | - Lina M Ismail
- Department of Biotechnology and Molecular Chemistry, Faculty of Science, Cairo University, Giza, Egypt
- Creative Egyptian Biotechnologists (CEB), Giza, Egypt
| | | | - Walid F Elkhatib
- Department of Microbiology and Immunology, Faculty of Pharmacy, Galala University, Suez, Egypt
- Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
| | - Heba Aboul-Ella
- Department of Pharmacognosy, Faculty of Pharmacy and Drug Technology, Egyptian Chinese University (ECU), Cairo, Egypt
- Scientific Research Group in Egypt (SRGE), Cairo, Egypt
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9
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Blazic M, Gautier C, Norberg T, Widersten M. High-throughput selection of (new) enzymes: phage display-mediated isolation of alkyl halide hydrolases from a library of active-site mutated epoxide hydrolases. Faraday Discuss 2024; 252:115-126. [PMID: 38828992 DOI: 10.1039/d4fd00001c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/05/2024]
Abstract
Epoxide hydrolase StEH1, from potato, is similar in overall structural fold and catalytic mechanism to haloalkane dehalogenase DhlA from Xanthobacter autotrophicus. StEH1 displays low (promiscuous) hydrolytic activity with (2-chloro)- and (2-bromo)ethanebenzene producing 2-phenylethanol. To investigate possibilities to amplify these very low dehalogenase activities, StEH1 was subjected to targeted randomized mutagenesis at five active-site amino acid residues and the resulting protein library was challenged for reactivity towards a bait chloride substrate. Enzymes catalyzing the first half-reaction of a hydrolytic cycle were isolated following monovalent phage display of the mutated proteins. Several StEH1 derived enzymes were identified with enhanced dehalogenase activities.
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Affiliation(s)
- Marija Blazic
- Department of Chemistry - BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
| | - Candice Gautier
- Department of Chemistry - BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
| | - Thomas Norberg
- Department of Chemistry - BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
| | - Mikael Widersten
- Department of Chemistry - BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
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10
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Abstract
A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.
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Affiliation(s)
- Christoph Rader
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida 33458, USA
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11
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Rakonjac J, Gold VAM, León-Quezada RI, Davenport CH. Structure, Biology, and Applications of Filamentous Bacteriophages. Cold Spring Harb Protoc 2024; 2024:pdb.over107754. [PMID: 37460152 DOI: 10.1101/pdb.over107754] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/03/2024]
Abstract
The closely related Escherichia coli Ff filamentous phages (f1, fd, and M13) have taken a fantastic journey over the past 60 years, from the urban sewerage from which they were first isolated, to their use in high-end technologies in multiple fields. Their relatively small genome size, high titers, and the virions that tolerate fusion proteins make the Ffs an ideal system for phage display. Folding of the fusions in the oxidizing environment of the E. coli periplasm makes the Ff phages a platform that allows display of eukaryotic surface and secreted proteins, including antibodies. Resistance of the Ffs to a broad range of pH and detergents facilitates affinity screening in phage display, whereas the stability of the virions at ambient temperature makes them suitable for applications in material science and nanotechnology. Among filamentous phages, only the Ffs have been used in phage display technology, because of the most advanced state of knowledge about their biology and the various tools developed for E. coli as a cloning host for them. Filamentous phages have been thought to be a rather small group, infecting mostly Gram-negative bacteria. A recent discovery of more than 10 thousand diverse filamentous phages in bacteria and archaea, however, opens a fascinating prospect for novel applications. The main aim of this review is to give detailed biological and structural information to researchers embarking on phage display projects. The secondary aim is to discuss the yet-unresolved puzzles, as well as recent developments in filamentous phage biology, from a viewpoint of their impact on current and future applications.
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Affiliation(s)
- Jasna Rakonjac
- School of Natural Sciences, Massey University, Auckland 0632, New Zealand
- Nanophage Technologies Ltd., Palmerston North, Manawatu 4474, New Zealand
| | - Vicki A M Gold
- Living Systems Institute University of Exeter, Exeter, EX4 4QD, United Kingdom
- Faculty of Health and Life Sciences, University of Exeter, Exeter, EX4 4QD, United Kingdom
| | - Rayén I León-Quezada
- School of Natural Sciences, Massey University, Auckland 0632, New Zealand
- Nanophage Technologies Ltd., Palmerston North, Manawatu 4474, New Zealand
| | - Catherine H Davenport
- School of Natural Sciences, Massey University, Auckland 0632, New Zealand
- Nanophage Technologies Ltd., Palmerston North, Manawatu 4474, New Zealand
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12
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Hutchings CJ, Sato AK. Phage display technology and its impact in the discovery of novel protein-based drugs. Expert Opin Drug Discov 2024; 19:887-915. [PMID: 39074492 DOI: 10.1080/17460441.2024.2367023] [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: 11/28/2023] [Accepted: 06/07/2024] [Indexed: 07/31/2024]
Abstract
INTRODUCTION Phage display technology is a well-established versatile in vitro display technology that has been used for over 35 years to identify peptides and antibodies for use as reagents and therapeutics, as well as exploring the diversity of alternative scaffolds as another option to conventional therapeutic antibody discovery. Such successes have been responsible for spawning a range of biotechnology companies, as well as many complementary technologies devised to expedite the drug discovery process and resolve bottlenecks in the discovery workflow. AREAS COVERED In this perspective, the authors summarize the application of phage display for drug discovery and provide examples of protein-based drugs that have either been approved or are being developed in the clinic. The amenability of phage display to generate functional protein molecules to challenging targets and recent developments of strategies and techniques designed to harness the power of sampling diverse repertoires are highlighted. EXPERT OPINION Phage display is now routinely combined with cutting-edge technologies to deep-mine antibody-based repertoires, peptide, or alternative scaffold libraries generating a wealth of data that can be leveraged, e.g. via artificial intelligence, to enable the potential for clinical success in the discovery and development of protein-based therapeutics.
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13
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Ruschig M, Nerlich J, Becker M, Meier D, Polten S, Cervantes-Luevano K, Kuhn P, Licea-Navarro AF, Hallermann S, Dübel S, Schubert M, Brown J, Hust M. Human antibodies neutralizing the alpha-latrotoxin of the European black widow. Front Immunol 2024; 15:1407398. [PMID: 38933276 PMCID: PMC11199383 DOI: 10.3389/fimmu.2024.1407398] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2024] [Accepted: 04/29/2024] [Indexed: 06/28/2024] Open
Abstract
Poisoning by widow-spider (genus Latrodectus) bites occurs worldwide. The illness, termed latrodectism, can cause severe and persistent pain and can lead to muscle rigidity, respiratory complications, and cardiac problems. It is a global health challenge especially in developing countries. Equine serum-derived polyclonal anti-sera are commercially available as a medication for patients with latrodectism, but the use of sera imposes potential inherent risks related to its animal origin. The treatment may cause allergic reactions in humans (serum sickness), including anaphylactic shock. Furthermore, equine-derived antivenom is observed to have batch-to-batch variability and poor specificity, as it is always an undefined mix of antibodies. Because latrodectism can be extremely painful but is rarely fatal, the use of antivenom is controversial and only a small fraction of patients is treated. In this work, recombinant human antibodies were selected against alpha-latrotoxin of the European black widow (Latrodectus tredecimguttatus) by phage display from a naïve antibody gene library. Alpha-Latrotoxin (α-LTX) binding scFv were recloned and produced as fully human IgG. A novel alamarBlue assay for venom neutralization was developed and used to select neutralizing IgGs. The human antibodies showed in vitro neutralization efficacy both as single antibodies and antibody combinations. This was also confirmed by electrophysiological measurements of neuronal activity in cell culture. The best neutralizing antibodies showed nanomolar affinities. Antibody MRU44-4-A1 showed outstanding neutralization efficacy and affinity to L. tredecimguttatus α-LTX. Interestingly, only two of the neutralizing antibodies showed cross-neutralization of the venom of the Southern black widow (Latrodectus mactans). This was unexpected, because in the current literature the alpha-latrotoxins are described as highly conserved. The here-engineered antibodies are candidates for future development as potential therapeutics and diagnostic tools, as they for the first time would provide unlimited supply of a chemically completely defined drug of constant quality and efficacy, which is also made without the use of animals.
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Affiliation(s)
- Maximilian Ruschig
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Jana Nerlich
- Faculty of Medicine, Carl-Ludwig-Institute of Physiology, Leipzig University, Leipzig, Germany
| | - Marlies Becker
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Doris Meier
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Saskia Polten
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Karla Cervantes-Luevano
- Departamento de Innovación Biomédica, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico
| | | | - Alexei Fedorovish Licea-Navarro
- Departamento de Innovación Biomédica, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico
| | - Stefan Hallermann
- Faculty of Medicine, Carl-Ludwig-Institute of Physiology, Leipzig University, Leipzig, Germany
| | - Stefan Dübel
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Maren Schubert
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
| | - Jeffrey Brown
- PETA Science Consortium International e.V., Stuttgart, Germany
| | - Michael Hust
- Departments of Biotechnology and Medical Biotechnology, Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
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14
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Aripov VS, Volkova NV, Ilyichev AA, Shcherbakov DN. Problems of creating antibody phage libraries and their solutions. Vavilovskii Zhurnal Genet Selektsii 2024; 28:249-257. [PMID: 38680186 PMCID: PMC11043502 DOI: 10.18699/vjgb-24-29] [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: 07/27/2023] [Revised: 01/18/2024] [Accepted: 01/19/2023] [Indexed: 05/01/2024] Open
Abstract
Phage display has become an efficient, reliable and popular molecular technique for generating libraries encompassing millions or even billions of clones of divergent peptides or proteins. The method is based on the correspondence between phage genotype and phenotype, which ensures the presentation of recombinant proteins of known amino acid composition on the surface of phage particles. The use of affinity selection allows one to choose variants with affinity for different targets from phage libraries. The implementation of the antibody phage display technique has revolutionized the field of clinical immunology, both for developing tools to diagnose infectious diseases and for producing therapeutic agents. It has also become the basis for efficient and relatively inexpensive methods for studying protein-protein interactions, receptor binding sites, as well as epitope and mimotope identification. The antibody phage display technique involves a number of steps, and the final result depends on their successful implementation. The diversity, whether natural or obtained by combinatorial chemistry, is the basis of any library. The choice of molecular techniques is critical to ensure that this diversity is maintained during the phage library preparation step and during the transformation of E. coli cells. After a helper phage is added to the suspension of transformed E. coli cells, a bacteriophage library is formed, which is a working tool for performing the affinity selection procedure and searching for individual molecules. Despite the apparent simplicity of generating phage antibody libraries, a number of subtleties need to be taken into account. First, there are the features of phage vector preparation. Currently, a large number of phagemid vectors have been developed, and their selection is also of great importance. The key step is preparing competent E. coli cells and the technology of their transformation. The choice of a helper phage and the method used to generate it is also important. This article discusses the key challenges faced by researchers in constructing phage antibody libraries.
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Affiliation(s)
- V S Aripov
- State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia
| | - N V Volkova
- State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia
| | - A A Ilyichev
- State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia
| | - D N Shcherbakov
- State Research Center of Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia
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15
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Zheng YY, Zhao L, Wei XF, Sun TZ, Xu FF, Wang GX, Zhu B. Vaccine Molecule Design Based on Phage Display and Computational Modeling against Rhabdovirus. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2024; 212:551-562. [PMID: 38197664 DOI: 10.4049/jimmunol.2300447] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Accepted: 12/12/2023] [Indexed: 01/11/2024]
Abstract
Rhabdoviruses with rich species lead a variety of high lethality and rapid transmission diseases to plants and animals around the globe. Vaccination is one of the most effective approaches to prevent and control virus disease. However, the key antigenic epitopes of glycoprotein being used for vaccine development are unclear. In this study, fish-derived Abs are employed for a Micropterus salmoides rhabdovirus (MSRV) vaccine design by phage display and bioinformatics analysis. We constructed an anti-MSRV phage Ab library to screen Abs for glycoprotein segment 2 (G2) (G129-266). Four M13-phage-displayed Abs (Ab-5, Ab-7, Ab-8 and Ab-30) exhibited strong specificity to target Ag, and Ab-7 had the highest affinity with MSRV. Ab-7 (300 μg/ml) significantly increased grass carp ovary cell viability to 83.40% and significantly decreased the titer of MSRV. Molecular docking results showed that the key region of Ag-Ab interaction was located in 10ESQEFTTLTSH20 of G2. G2Ser11 and G2Gln12 were replaced with alanine, respectively, and molecular docking results showed that the Ag-Ab was nonbinding (ΔG > 0). Then, the peptide vaccine KLH-G210-20 was immunized to M. salmoides via i.p. injection. ELISA result showed that the serum Ab potency level increased significantly (p < 0.01). More importantly, the challenge test demonstrated that the peptide vaccine elicited robust protection against MSRV invasion, and the relative percentage survival reached 62.07%. Overall, this study proposed an approach for screening key epitope by combining phage display technology and bioinformatics tools to provide a reliable theoretical reference for the prevention and control of viral diseases.
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Affiliation(s)
- Yu-Ying Zheng
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Liang Zhao
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Xue-Feng Wei
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Tian-Zi Sun
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Fei-Fan Xu
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Gao-Xue Wang
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
- Key Laboratory of Livestock Biology, Northwest A&F University, Yangling, Shaanxi, China
| | - Bin Zhu
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
- Key Laboratory of Livestock Biology, Northwest A&F University, Yangling, Shaanxi, China
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16
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Irvine EB, Reddy ST. Advancing Antibody Engineering through Synthetic Evolution and Machine Learning. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2024; 212:235-243. [PMID: 38166249 DOI: 10.4049/jimmunol.2300492] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Accepted: 10/20/2023] [Indexed: 01/04/2024]
Abstract
Abs are versatile molecules with the potential to achieve exceptional binding to target Ags, while also possessing biophysical properties suitable for therapeutic drug development. Protein display and directed evolution systems have transformed synthetic Ab discovery, engineering, and optimization, vastly expanding the number of Ab clones able to be experimentally screened for binding. Moreover, the burgeoning integration of high-throughput screening, deep sequencing, and machine learning has further augmented in vitro Ab optimization, promising to accelerate the design process and massively expand the Ab sequence space interrogated. In this Brief Review, we discuss the experimental and computational tools employed in synthetic Ab engineering and optimization. We also explore the therapeutic challenges posed by developing Abs for infectious diseases, and the prospects for leveraging machine learning-guided protein engineering to prospectively design Abs resistant to viral escape.
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Affiliation(s)
- Edward B Irvine
- Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
| | - Sai T Reddy
- Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
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17
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Dübel S. Can antibodies be "vegan"? A guide through the maze of today's antibody generation methods. MAbs 2024; 16:2343499. [PMID: 38634488 PMCID: PMC11028021 DOI: 10.1080/19420862.2024.2343499] [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: 02/28/2024] [Accepted: 04/11/2024] [Indexed: 04/19/2024] Open
Abstract
There is no doubt that today's life sciences would look very different without the availability of millions of research antibody products. Nevertheless, the use of antibody reagents that are poorly characterized has led to the publication of false or misleading results. The use of laboratory animals to produce research antibodies has also been criticized. Surprisingly, both problems can be addressed with the same technology. This review charts today's maze of different antibody formats and the various methods for antibody production and their interconnections, ultimately concluding that sequence-defined recombinant antibodies offer a clear path to both improved quality of experimental data and reduced use of animals.
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Affiliation(s)
- Stefan Dübel
- Institute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany
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18
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Islam MS, Fan J, Pan F. The power of phages: revolutionizing cancer treatment. Front Oncol 2023; 13:1290296. [PMID: 38033486 PMCID: PMC10684691 DOI: 10.3389/fonc.2023.1290296] [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: 09/07/2023] [Accepted: 10/31/2023] [Indexed: 12/02/2023] Open
Abstract
Cancer is a devastating disease with a high global mortality rate and is projected to increase further in the coming years. Current treatment options, such as chemotherapy and radiation therapy, have limitations including side effects, variable effectiveness, high costs, and limited availability. There is a growing need for alternative treatments that can target cancer cells specifically with fewer side effects. Phages, that infect bacteria but not eukaryotic cells, have emerged as promising cancer therapeutics due to their unique properties, including specificity and ease of genetic modification. Engineered phages can transform cancer treatment by targeting cancer cells while sparing healthy ones. Phages exhibit versatility as nanocarriers, capable of delivering therapeutic agents like gene therapy, immunotherapy, and vaccines. Phages are extensively used in vaccine development, with filamentous, tailed, and icosahedral phages explored for different antigen expression possibilities. Engineered filamentous phages bring benefits such as built in adjuvant properties, cost-effectiveness, versatility in multivalent formulations, feasibility of oral administration, and stability. Phage-based vaccines stimulate the innate immune system by engaging pattern recognition receptors on antigen-presenting cells, enhancing phage peptide antigen presentation to B-cells and T-cells. This review presents recent phage therapy advances and challenges in cancer therapy, exploring its versatile tools and vaccine potential.
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Affiliation(s)
- Md. Sharifull Islam
- Center for Cancer Immunology, Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Jie Fan
- Department of Cardiology, Handan Central Hospital, Handan, Hebei, China
| | - Fan Pan
- Center for Cancer Immunology, Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
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19
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Najafi A, Valadan R, Asgarian-Omran H, Rafiei A, Tehrani M. Single domain antibodies specific for HER2 dimerization domain effectively disrupts HER2 dimerization. Int Immunopharmacol 2023; 124:110999. [PMID: 37804659 DOI: 10.1016/j.intimp.2023.110999] [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: 08/09/2023] [Revised: 09/23/2023] [Accepted: 09/24/2023] [Indexed: 10/09/2023]
Abstract
Dimer-dependent phosphorylation of HER2 receptor is a key event for the signal transduction of HER family of receptors which correlates with tumor invasion and metastasis. New generation of therapies based on dimerization domain inhibition using monoclonal or fragment antibodies was introduced. A potent method for manufacturing antibodies and antibody fragments is the phage display antibody library method. A recombinant phage was generated using the phage display method from synthetic dAb library. Subtractive biopanning was performed on sepharose 4b resin. Evaluation of success of subtractive biopanning was confirmed by the PCR fingerprinting after the fourth round of biopanning. The fourth round of biopanning results in the isolation of several dimerization domain reactive clones based on the polyclonal phage ELISA results. Monoclonal phage cell ELISA was used to select the positive clones with the highest affinity, and they were subsequently employed for functional tests. Cell-ELISA, MTT assay and dimerization inhibition test revealed that the reactivity and specificity of the selected monoclonal phage to dimerization domain of HER2. Further, Annexin V/PI staining and gene expression analysis showed that increased apoptosis rates. Also, in silico binding of the selected clones to conformational structure of HER2 was applied, using protein-protein docking tool of the ICM-Pro software, and showed sdAbs were specifically interacted with dimerization domain of the receptor. In conclusion, we have identified a single domain targeting HER2 dimerization, which represents a promising therapeutic and diagnostic candidate for HER2-positive cancers. Purified sdAb needs to more research to evaluate it both in vivo and in vitro via functional tests to determine if it can be applied for treatment and diagnostics.
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Affiliation(s)
- Ahmad Najafi
- Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
| | - Reza Valadan
- Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
| | - Hossein Asgarian-Omran
- Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
| | - Alireza Rafiei
- Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
| | - Mohsen Tehrani
- Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
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20
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Chang FL, Tsai KC, Lin TY, Chiang CW, Pan SL, Lee YC. Effectiveness of anti-erythropoietin producing Hepatocellular receptor Type-A2 antibody in pancreatic cancer treatment. Heliyon 2023; 9:e21774. [PMID: 38034633 PMCID: PMC10682614 DOI: 10.1016/j.heliyon.2023.e21774] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 10/23/2023] [Accepted: 10/27/2023] [Indexed: 12/02/2023] Open
Abstract
Erythropoietin-producing hepatocyte receptor type A2 (EphA2) is a tyrosine kinase that binds to ephrins (e.g., ephrin-A1) to initiate bidirectional signaling between cells. The binding of EphA2 and ephrin-A1 leads to the inhibition of Ras-MAPK activity and tumor growth. During tumorigenesis, the normal interaction between EphA2 and ephrin-A1 is hindered, which leads to the overexpression of EphA2 and induces cancer. The overexpression of EphA2 has been identified as a notable tumor marker in diagnosing and treating pancreatic cancer. In this study, we used phage display to isolate specific antibodies against the active site of EphA2 by using a discontinuous recombinant epitope for immunization. The therapeutic efficacy and inhibition mechanism of the generated antibody against pancreatic cancer was validated and clarified. The generated antibodies were bound to the conformational epitope of endogenous EphA2 on cancer cells, thus inducing cellular endocytosis and causing EphA2 degradation. Molecule signals pAKT, pERK, pFAK, and pSTAT3 were weakened, inhibiting the proliferation and migration of pancreatic cancer cells. The humanized antibody hSD5 could effectively inhibit the growth of the xenograft pancreatic cancer tumor cells BxPc-3 and Mia PaCa-2 in mice, respectively. When antibody hSD5 was administered with gemcitabine, significantly improved effects on tumor growth inhibition were observed. Based on the efficacy of the IgG hSD5 antibodies, clinical administration of the hSD5 antibodies is likely to suppress tumors in patients with pancreatic cancer and abnormal activation or overexpression of EphA2 signaling.
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Affiliation(s)
- Fu-Ling Chang
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan
| | - Keng-Chang Tsai
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Tsai-Yu Lin
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Chen-Wei Chiang
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Shiow-Lin Pan
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
- Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- TMU Research Center for Drug Discovery, Taipei Medical University, Taipei, Taiwan
| | - Yu-Ching Lee
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
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21
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França RKA, Studart IC, Bezerra MRL, Pontes LQ, Barbosa AMA, Brigido MM, Furtado GP, Maranhão AQ. Progress on Phage Display Technology: Tailoring Antibodies for Cancer Immunotherapy. Viruses 2023; 15:1903. [PMID: 37766309 PMCID: PMC10536222 DOI: 10.3390/v15091903] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Revised: 08/31/2023] [Accepted: 09/05/2023] [Indexed: 09/29/2023] Open
Abstract
The search for innovative anti-cancer drugs remains a challenge. Over the past three decades, antibodies have emerged as an essential asset in successful cancer therapy. The major obstacle in developing anti-cancer antibodies is the need for non-immunogenic antibodies against human antigens. This unique requirement highlights a disadvantage to using traditional hybridoma technology and thus demands alternative approaches, such as humanizing murine monoclonal antibodies. To overcome these hurdles, human monoclonal antibodies can be obtained directly from Phage Display libraries, a groundbreaking tool for antibody selection. These libraries consist of genetically engineered viruses, or phages, which can exhibit antibody fragments, such as scFv or Fab on their capsid. This innovation allows the in vitro selection of novel molecules directed towards cancer antigens. As foreseen when Phage Display was first described, nowadays, several Phage Display-derived antibodies have entered clinical settings or are undergoing clinical evaluation. This comprehensive review unveils the remarkable progress in this field and the possibilities of using clever strategies for phage selection and tailoring the refinement of antibodies aimed at increasingly specific targets. Moreover, the use of selected antibodies in cutting-edge formats is discussed, such as CAR (chimeric antigen receptor) in CAR T-cell therapy or ADC (antibody drug conjugate), amplifying the spectrum of potential therapeutic avenues.
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Affiliation(s)
- Renato Kaylan Alves França
- Molecular Immunology Laboratory, Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, Brasilia 70910-900, Brazil; (R.K.A.F.); (M.M.B.)
- Graduate Program in Molecular Pathology, University of Brasilia, Brasilia 70910-900, Brazil
| | - Igor Cabral Studart
- Oswaldo Cruz Foundation, Fiocruz Ceará, Eusébio 61773-270, Brazil; (I.C.S.); (M.R.L.B.); (L.Q.P.); (A.M.A.B.); (G.P.F.)
- Graduate Program in Biotechnology of Natural Resources, Federal University of Ceará, Fortaleza 60440-970, Brazil
| | - Marcus Rafael Lobo Bezerra
- Oswaldo Cruz Foundation, Fiocruz Ceará, Eusébio 61773-270, Brazil; (I.C.S.); (M.R.L.B.); (L.Q.P.); (A.M.A.B.); (G.P.F.)
- Graduate Program in Biotechnology of Natural Resources, Federal University of Ceará, Fortaleza 60440-970, Brazil
| | - Larissa Queiroz Pontes
- Oswaldo Cruz Foundation, Fiocruz Ceará, Eusébio 61773-270, Brazil; (I.C.S.); (M.R.L.B.); (L.Q.P.); (A.M.A.B.); (G.P.F.)
- Graduate Program in Biotechnology of Natural Resources, Federal University of Ceará, Fortaleza 60440-970, Brazil
| | - Antonio Marcos Aires Barbosa
- Oswaldo Cruz Foundation, Fiocruz Ceará, Eusébio 61773-270, Brazil; (I.C.S.); (M.R.L.B.); (L.Q.P.); (A.M.A.B.); (G.P.F.)
- Graduate Program in Applied Informatics, University of Fortaleza, Fortaleza 60811-905, Brazil
| | - Marcelo Macedo Brigido
- Molecular Immunology Laboratory, Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, Brasilia 70910-900, Brazil; (R.K.A.F.); (M.M.B.)
| | - Gilvan Pessoa Furtado
- Oswaldo Cruz Foundation, Fiocruz Ceará, Eusébio 61773-270, Brazil; (I.C.S.); (M.R.L.B.); (L.Q.P.); (A.M.A.B.); (G.P.F.)
- Graduate Program in Biotechnology of Natural Resources, Federal University of Ceará, Fortaleza 60440-970, Brazil
| | - Andréa Queiroz Maranhão
- Molecular Immunology Laboratory, Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, Brasilia 70910-900, Brazil; (R.K.A.F.); (M.M.B.)
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22
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Jia Q, Xiang Y. Cryo-EM structure of a bacteriophage M13 mini variant. Nat Commun 2023; 14:5421. [PMID: 37669979 PMCID: PMC10480500 DOI: 10.1038/s41467-023-41151-7] [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: 03/28/2023] [Accepted: 08/24/2023] [Indexed: 09/07/2023] Open
Abstract
Filamentous bacteriophages package their circular, single stranded DNA genome with the major coat protein pVIII and the minor coat proteins pIII, pVII, pVI, and pIX. Here, we report the cryo-EM structure of a ~500 Å long bacteriophage M13 mini variant. The distal ends of the mini phage are sealed by two cap-like complexes composed of the minor coat proteins. The top cap complex consists of pVII and pIX, both exhibiting a single helix structure. Arg33 of pVII and Glu29 of pIX, located on the inner surface of the cap, play a key role in recognizing the genome packaging signal. The bottom cap complex is formed by the hook-like structures of pIII and pVI, arranged in helix barrels. Most of the inner ssDNA genome adopts a double helix structure with a similar pitch to that of the A-form double-stranded DNA. These findings provide insights into the assembly of filamentous bacteriophages.
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Affiliation(s)
- Qi Jia
- Beijing Frontier Research Center for Biological Structure, Center for Infectious Disease Research, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, P.R. China
| | - Ye Xiang
- Beijing Frontier Research Center for Biological Structure, Center for Infectious Disease Research, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, P.R. China.
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23
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Li A, Swanson M, Sullivan N, Homan Y, Nahas D, Mukhopadhyay S, Li HH, Cao Y, Xu W, Tang H, Vora KA, Chen Z. Phage-derived anti-idiotype and anti-YTE antibodies in development of MK-1654 pharmacokinetic and immune response assays. Bioanalysis 2023; 15:1049-1067. [PMID: 37515532 DOI: 10.4155/bio-2023-0081] [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] [Indexed: 07/31/2023] Open
Abstract
Background: MK-1654 is a fully human monoclonal antibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodies can deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.
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Affiliation(s)
- April Li
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
| | - Michael Swanson
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
- Current address: Janssen Pharmaceutical, Ambler, PA 19002, USA
| | - Nicole Sullivan
- Infectious Diseases and Vaccine Research, Merck and Co., Inc., West Point, PA 19486, USA
| | - Ying Homan
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
| | - Debbie Nahas
- Infectious Diseases and Vaccine Research, Merck and Co., Inc., West Point, PA 19486, USA
| | - Shreya Mukhopadhyay
- Infectious Diseases and Vaccine Research, Merck and Co., Inc., West Point, PA 19486, USA
| | - Hualin Helen Li
- Analytical Research and Development, Merck and Co., Inc., West Point, PA 19486, USA
| | - Yu Cao
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
| | - Weifeng Xu
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
| | - Huaping Tang
- PCD Regulated Immunogenicity and Molecular, Merck and Co., Inc., West Point, PA 19486, USA
- Current address: GSK Pharmaceutical, Collegeville, PA 19426, USA
| | - Kalpit A Vora
- Infectious Diseases and Vaccine Research, Merck and Co., Inc., West Point, PA 19486, USA
| | - Zhifeng Chen
- Infectious Diseases and Vaccine Research, Merck and Co., Inc., West Point, PA 19486, USA
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24
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Zhu C, Xu L, Chen L, Zhang Z, Zhang Y, Wu W, Li C, Liu S, Xiang S, Dai S, Zhang J, Guo H, Zhou Y, Wang F. Epitope-Directed Antibody Elicitation by Genetically Encoded Chemical Cross-Linking Reactivity in the Antigen. ACS CENTRAL SCIENCE 2023; 9:1229-1240. [PMID: 37396855 PMCID: PMC10311653 DOI: 10.1021/acscentsci.3c00265] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Indexed: 07/04/2023]
Abstract
No current methods can selectively elicit an antibody response to a specific conformational epitope in a whole antigen in vivo. Here, we incorporated Nε-acryloyl-l-lysine (AcrK) or Nε-crotonyl-l-lysine (Kcr) with cross-linking activities into the specific epitopes of antigens and immunized mice to generate antibodies that can covalently cross-link with the antigens. By taking advantage of antibody clonal selection and evolution in vivo, an orthogonal antibody-antigen cross-linking reaction can be generated. With this mechanism, we developed a new approach for facile elicitation of antibodies binding to specific epitopes of the antigen in vivo. Antibody responses were directed and enriched to the target epitopes on protein antigens or peptide-KLH conjugates after mouse immunization with the AcrK or Kcr-incorporated immunogens. The effect is so prominent that the majority of selected hits bind to the target epitope. Furthermore, the epitope-specific antibodies effectively block IL-1β from activating its receptor, indicating its potential for the development of protein subunit vaccines.
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Affiliation(s)
- Chaoyang Zhu
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Liang Xu
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Longxin Chen
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- Molecular
Biology Laboratory, Zhengzhou Normal University, Zhengzhou 450044, China
| | - Zihan Zhang
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Yuhan Zhang
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Weiping Wu
- Suzhou
Institute for Biomedical Research, Suzhou, Jiangsu 215028, China
| | - Chengxiang Li
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Shuang Liu
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Shuqin Xiang
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Shengwang Dai
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- College
of Life Sciences, University of Chinese
Academy of Sciences, Beijing 100101, China
| | - Jay Zhang
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- Suzhou
Institute for Biomedical Research, Suzhou, Jiangsu 215028, China
| | - Hui Guo
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- Suzhou
Institute for Biomedical Research, Suzhou, Jiangsu 215028, China
- Beijing
Translational Center for Biopharmaceuticals, Beijing 100101, China
| | - Yinjian Zhou
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- Beijing
Translational Center for Biopharmaceuticals, Beijing 100101, China
| | - Feng Wang
- Key
Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- Suzhou
Institute for Biomedical Research, Suzhou, Jiangsu 215028, China
- Beijing
Translational Center for Biopharmaceuticals, Beijing 100101, China
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25
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Kumari S, Singh K, Singh N, Khan S, Kumar A. Phage display and human disease detection. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 201:151-172. [PMID: 37770169 DOI: 10.1016/bs.pmbts.2023.03.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/03/2023]
Abstract
Phage display is a significant and active molecular method and has continued crucial for investigative sector meanwhile its unearthing in 1985. This practice has numerous benefits: the association among physiology and genome, the massive variety of variant proteins showed in sole collection and the elasticity of collection that can be achieved. It suggests a diversity of stages for manipulating antigen attachment; yet, variety and steadiness of exhibited library are an alarm. Additional improvements, like accumulation of non-canonical amino acids, resulting in extension of ligands that can be recognized through collection, will support in expansion of the probable uses and possibilities of technology. Epidemic of COVID-19 had taken countless lives, and while indicative prescriptions were provided to diseased individuals, still no prevention was observed for the contamination. Phage demonstration has presented an in-depth understanding into protein connections included in pathogenesis. Phage display knowledge is developing as an influential, inexpensive, quick, and effectual method to grow novel mediators for the molecular imaging and analysis of cancer.
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Affiliation(s)
- Sonu Kumari
- Department of Biotechnology, Faculty of Engineering and Technology, Rama University, Kanpur, Uttar Pradesh, India
| | - Krati Singh
- Department of Biotechnology, Banasthali University, Newai, Rajasthan, India
| | - Neha Singh
- Department of Biotechnology, Banasthali University, Newai, Rajasthan, India
| | - Suphiya Khan
- Department of Biotechnology, Banasthali University, Newai, Rajasthan, India
| | - Ajay Kumar
- Department of Biotechnology, Faculty of Engineering and Technology, Rama University, Kanpur, Uttar Pradesh, India.
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26
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Cyr MG, Wilson HD, Spierling AL, Chang J, Peng H, Steinberger P, Rader C. Concerted Antibody and Antigen Discovery by Differential Whole-cell Phage Display Selections and Multi-omic Target Deconvolution. J Mol Biol 2023; 435:168085. [PMID: 37019174 PMCID: PMC10148915 DOI: 10.1016/j.jmb.2023.168085] [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: 01/31/2023] [Revised: 03/24/2023] [Accepted: 03/28/2023] [Indexed: 04/05/2023]
Abstract
Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.
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Affiliation(s)
- Matthew G Cyr
- Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL, USA; Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA. https://twitter.com/CyrialDilutions
| | - Henry D Wilson
- Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL, USA
| | - Anna-Lena Spierling
- Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA
| | - Jing Chang
- Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA
| | - Haiyong Peng
- Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA
| | - Peter Steinberger
- Institute of Immunology, Medical University of Vienna, Vienna, Austria
| | - Christoph Rader
- Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL, USA; Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA.
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27
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Lee YC, Lai GH, Lin TY, Tseng TS, Tsai TH, Chen WC, Lee CC, Tsai KC. Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing. J Biol Eng 2023; 17:30. [PMID: 37095503 PMCID: PMC10127376 DOI: 10.1186/s13036-023-00350-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Accepted: 04/17/2023] [Indexed: 04/26/2023] Open
Abstract
BACKGROUND The main commercially available methods for detecting small molecules of mycotoxins in traditional Chinese medicine (TCM) and functional foods are enzyme-linked immunosorbent assay and mass spectrometry. Regarding the development of diagnostic antibody reagents, effective methods for the rapid preparation of specific monoclonal antibodies are inadequate. METHODS In this study, a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library with a glove-like cavity configuration was established using phage display technology in synthetic biology. We applied this unique SynaGG library on the small molecule aflatoxin B1 (AFB1), which has strong hepatotoxicity, to isolate specific nanobodies with high affinity for AFB1. RESULT These nanobodies exhibit no cross-reactivity with the hapten methotrexate, which is recognized by the original antibody template. By binding to AFB1, two nanobodies can neutralize AFB1-induced hepatocyte growth inhibition. Using molecular docking, we found that the unique non-hypervariable complementarity-determining region 4 (CDR4) loop region of the nanobody was involved in the interaction with AFB1. Specifically, the CDR4's positively charged amino acid arginine directed the binding interaction between the nanobody and AFB1. We then rationally optimized the interaction between AFB1 and the nanobody by mutating serine at position 2 into valine. The binding affinity of the nanobody to AFB1 was effectively improved, and this result supported the use of molecular structure simulation for antibody optimization. CONCLUSION In summary, this study revealed that the novel SynaGG library, which was constructed through computer-aided design, can be used to isolate nanobodies that specifically bind to small molecules. The results of this study could facilitate the development of nanobody materials to detect small molecules for the rapid screening of TCM materials and foods in the future.
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Affiliation(s)
- Yu-Ching Lee
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Gar-Hwa Lai
- Department of Orthopedics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
| | - Tsai-Yu Lin
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Tien-Sheng Tseng
- Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan
| | - Tsung-Hsun Tsai
- Department of Psychiatry, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan
| | - Wang-Chuan Chen
- The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan
- Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan
| | - Cheng-Chung Lee
- The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Keng-Chang Tsai
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, No. 155-1, Sec. 2, Linong St., Beitou District, Taipei, 11221, Taiwan.
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28
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Winton AJ, Allen MA. Rational Design of a Bifunctional Peptide Exhibiting Lithium Titanate Oxide and Carbon Nanotube Affinities for Lithium-Ion Battery Applications. ACS APPLIED MATERIALS & INTERFACES 2023; 15:8579-8589. [PMID: 36729082 DOI: 10.1021/acsami.2c18018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
Phage display is employed as a method for identifying polypeptides that bind to lithium-ion battery materials, specifically lithium titanate oxide (LTO) and multiwalled carbon nanotubes (MWCNTs). Output/input assays are used as a quantitative measure to narrow down the strongest binding polypeptides from several peptides selected through biopanning. Negatively stained transmission electron microscopy is used to verify that a phage presenting a particular LTO or MWCNT binding peptide sequence colocalizes with the respective material. Heterologous expression allows for ample polypeptides to be grown and purified using a peptide expression vector. Isothermal titration calorimetry in conjunction with alanine scanning enables determination of the pertinent residues involved in LTO binding and yields a dissociation constant of 3.41 μM. A rationally designed bifunctional peptide exhibiting LTO and MWCNT binding domains is subsequently validated to exhibit both LTO and MWCNT affinities and is incorporated as a binding agent in LTO coin-type electrochemical cells where the bifunctional peptide demonstrates stability at high cycle rates and potential as an alternative to non-specific binding agents for aqueous slurry processing of lithium-ion battery electrodes.
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Affiliation(s)
- Alexander J Winton
- Department of Chemistry & Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, United States
| | - Mark A Allen
- Department of Chemistry & Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, United States
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29
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Yang Z, Wu Z, Santich BH, Liu J, Liu C, Cheung NKV. Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach. Methods Mol Biol 2023; 2702:327-345. [PMID: 37679628 DOI: 10.1007/978-1-0716-3381-6_17] [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] [Indexed: 09/09/2023]
Abstract
Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T cell biology but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B cell epitope space on the surface of the pMHC complex (typical peptide MW = 1 kDa versus MHC MW = 45 kDa) and to the multiple efficiency limiting steps inherent in these methods. In this chapter we describe phage display approaches, including a cell panning strategy, for the rapid generation of such antibodies with high specificity and affinity.
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Affiliation(s)
| | - Zhihao Wu
- Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Brian H Santich
- Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
| | | | - Cheng Liu
- Eureka Therapeutics, Emeryville, CA, USA
| | - Nai-Kong V Cheung
- Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
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30
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Nur A, Schubert M, Lai JY, Hust M, Choong YS, Isa WYHW, Lim TS. Antibody Phage Display. Methods Mol Biol 2023; 2702:3-12. [PMID: 37679612 DOI: 10.1007/978-1-0716-3381-6_1] [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] [Indexed: 09/09/2023]
Abstract
The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.
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Affiliation(s)
- Alia Nur
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
| | - Maren Schubert
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Jing Yi Lai
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
| | - Michael Hust
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Yee Siew Choong
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
| | - Wan Yus Haniff Wan Isa
- School of Medical Sciences, Department of Medicine, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
| | - Theam Soon Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia.
- Analytical Biochemistry Research Center, Universiti Sains Malaysia, Penang, Malaysia.
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Nguyen TTH, Lee JS, Shim H. Construction of Rabbit Immune Antibody Libraries. Methods Mol Biol 2023; 2702:93-106. [PMID: 37679617 DOI: 10.1007/978-1-0716-3381-6_6] [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] [Indexed: 09/09/2023]
Abstract
Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.
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Affiliation(s)
| | | | - Hyunbo Shim
- Department of Life Sciences, Ewha Womans Univesity, Seoul, Korea.
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Ruschig M, Heine PA, Fühner V, Zilkens KJK, Steinke S, Schubert M, Bertoglio F, Hust M. Construction of Human Immune and Naive scFv Phage Display Libraries. Methods Mol Biol 2023; 2702:15-37. [PMID: 37679613 DOI: 10.1007/978-1-0716-3381-6_2] [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] [Indexed: 09/09/2023]
Abstract
Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.
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Affiliation(s)
- Maximilian Ruschig
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Philip Alexander Heine
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Viola Fühner
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | | | - Stephan Steinke
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Maren Schubert
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Federico Bertoglio
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
- Choose Life Biotech SA, Bellinzona, Switzerland
| | - Michael Hust
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany.
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Gao X, Fan L, Zheng B, Li H, Wang J, Zhang L, Li J, Zhu F. Binding and neutralizing abilities of antibodies towards SARS-CoV-2 S2 domain. Hum Vaccin Immunother 2022; 18:2055373. [PMID: 35417303 PMCID: PMC9225664 DOI: 10.1080/21645515.2022.2055373] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Accepted: 03/15/2022] [Indexed: 12/04/2022] Open
Abstract
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants have been reported to be resistant to several neutralizing antibodies (NAbs) targeting Receptor Binding Domain (RBD) and N Terminal Domain (NTD) of spike (S) protein and thus inducing immune escape. However, fewer studies were carried out to investigate the neutralizing ability of S2-specific antibodies. In this research, 10 monoclonal antibodies (mAbs) targeting SARS-CoV-2 S2 subunit were generated from Coronavirus Disease 2019 (COVID-19) convalescent patients by phage display technology and molecular cloning technology. The binding activity of these S2-mAbs toward SARS-CoV-2 S, SARS-CoV-2 S2, SARS-CoV-2 RBD, SARS-CoV-2 NTD, severe acute respiratory syndrome coronavirus (SARS-CoV) S, SARS-CoV S2 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) S proteins were evaluated by enzyme-linked immunosorbent assay (ELISA). Their neutralizing potency toward SARS-CoV-2 wild-type (WT), B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.1 and B.1.621 variants were determined by pseudo-virus-based neutralization assay. Results showed that S2E7-mAb had cross-activity to S or S2 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, while with limited neutralizing activity to pseudo-viruses of SARS-CoV-2 WT and variants. It is undeniable that the binding and neutralizing activities of the S2-targeting mAbs are significantly weaker than the previously reported antibodies targeting RBD and NTD, but our study may provide some evidences for understanding immune protection and identifying targets for vaccine design based on the conserved S2 subunit.
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Affiliation(s)
- Xingsu Gao
- Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, PR China
| | - Linlin Fan
- Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, PR China
| | - Binyang Zheng
- Vaccine Clinical Evaluation Department, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, PR China
| | - Haoze Li
- Vazyme Biotech Co, Ltd., Nanjing, PR China
| | - Jiwei Wang
- Vazyme Biotech Co, Ltd., Nanjing, PR China
| | - Li Zhang
- Vaccine Clinical Evaluation Department, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, PR China
| | - Jingxin Li
- Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, PR China
- Vaccine Clinical Evaluation Department, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, PR China
- Institute of Global Public Health and Emergency Pharmacy, China Pharmaceutical University, Nanjing, PR China
| | - Fengcai Zhu
- Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, PR China
- Institute of Global Public Health and Emergency Pharmacy, China Pharmaceutical University, Nanjing, PR China
- NHC Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, PR China
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Wang Z, Wang G, Lu H, Li H, Tang M, Tong A. Development of therapeutic antibodies for the treatment of diseases. MOLECULAR BIOMEDICINE 2022; 3:35. [PMID: 36418786 PMCID: PMC9684400 DOI: 10.1186/s43556-022-00100-4] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Accepted: 10/24/2022] [Indexed: 11/25/2022] Open
Abstract
Since the first monoclonal antibody drug, muromonab-CD3, was approved for marketing in 1986, 165 antibody drugs have been approved or are under regulatory review worldwide. With the approval of new drugs for treating a wide range of diseases, including cancer and autoimmune and metabolic disorders, the therapeutic antibody drug market has experienced explosive growth. Monoclonal antibodies have been sought after by many biopharmaceutical companies and scientific research institutes due to their high specificity, strong targeting abilities, low toxicity, side effects, and high development success rate. The related industries and markets are growing rapidly, and therapeutic antibodies are one of the most important research and development areas in the field of biology and medicine. In recent years, great progress has been made in the key technologies and theoretical innovations provided by therapeutic antibodies, including antibody-drug conjugates, antibody-conjugated nuclides, bispecific antibodies, nanobodies, and other antibody analogs. Additionally, therapeutic antibodies can be combined with technologies used in other fields to create new cross-fields, such as chimeric antigen receptor T cells (CAR-T), CAR-natural killer cells (CAR-NK), and other cell therapy. This review summarizes the latest approved or in regulatory review therapeutic antibodies that have been approved or that are under regulatory review worldwide, as well as clinical research on these approaches and their development, and outlines antibody discovery strategies that have emerged during the development of therapeutic antibodies, such as hybridoma technology, phage display, preparation of fully human antibody from transgenic mice, single B-cell antibody technology, and artificial intelligence-assisted antibody discovery.
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Affiliation(s)
- Zeng Wang
- State Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Guoqing Wang
- Department of Neurosurgery, West China Medical School, West China Hospital, Sichuan University, Chengdu, China
| | - Huaqing Lu
- State Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Hongjian Li
- Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China
| | - Mei Tang
- State Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Aiping Tong
- State Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
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35
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Chen Z, Zhang P, Matsuoka Y, Tsybovsky Y, West K, Santos C, Boyd LF, Nguyen H, Pomerenke A, Stephens T, Olia AS, Zhang B, De Giorgi V, Holbrook MR, Gross R, Postnikova E, Garza NL, Johnson RF, Margulies DH, Kwong PD, Alter HJ, Buchholz UJ, Lusso P, Farci P. Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants. Cell Rep 2022; 41:111528. [PMID: 36302375 PMCID: PMC9554601 DOI: 10.1016/j.celrep.2022.111528] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Revised: 07/29/2022] [Accepted: 09/28/2022] [Indexed: 11/17/2022] Open
Abstract
The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.
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Affiliation(s)
- Zhaochun Chen
- Hepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
| | - Peng Zhang
- Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Yumiko Matsuoka
- RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Yaroslav Tsybovsky
- Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Kamille West
- Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD, USA
| | - Celia Santos
- RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Lisa F Boyd
- Molecular Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Hanh Nguyen
- Hepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Anna Pomerenke
- Hepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Tyler Stephens
- Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Adam S Olia
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Baoshan Zhang
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Valeria De Giorgi
- Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD, USA
| | - Michael R Holbrook
- National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility, National Institutes of Health, Frederick, MD, USA
| | - Robin Gross
- National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility, National Institutes of Health, Frederick, MD, USA
| | - Elena Postnikova
- National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility, National Institutes of Health, Frederick, MD, USA
| | - Nicole L Garza
- SARS-CoV-2 Virology Core, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Reed F Johnson
- SARS-CoV-2 Virology Core, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - David H Margulies
- Molecular Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Peter D Kwong
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Harvey J Alter
- Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD, USA
| | - Ursula J Buchholz
- RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Paolo Lusso
- Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Patrizia Farci
- Hepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
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André AS, Moutinho I, Dias JNR, Aires-da-Silva F. In vivo Phage Display: A promising selection strategy for the improvement of antibody targeting and drug delivery properties. Front Microbiol 2022; 13:962124. [PMID: 36225354 PMCID: PMC9549074 DOI: 10.3389/fmicb.2022.962124] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2022] [Accepted: 09/01/2022] [Indexed: 11/13/2022] Open
Abstract
The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity responses have led to the development of alternative approaches for the streamlined identification of most effective antibodies. Within this context, display selection technologies such as phage display, ribosome display, yeast display, bacterial display, and mammalian cell surface display have been widely promoted over the past three decades as ideal alternatives to traditional hybridoma methods. The display of antibodies on phages is probably the most widespread and powerful of these methods and, since its invention in late 1980s, significant technological advancements in the design, construction, and selection of antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. With evolving novel disease targets and the emerging of a new generation of therapeutic antibodies, such as bispecific antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapies, it is clear that phage display is expected to continue to play a central role in antibody development. Nevertheless, for non-standard and more demanding cases aiming to generate best-in-class therapeutic antibodies against challenging targets and unmet medical needs, in vivo phage display selections by which phage libraries are directly injected into animals or humans for isolating and identifying the phages bound to specific tissues offer an advantage over conventional in vitro phage display screening procedures. Thus, in the present review, we will first summarize a general overview of the antibody therapeutic market, the different types of antibody fragments, and novel engineered variants that have already been explored. Then, we will discuss the state-of-the-art of in vivo phage display methodologies as a promising emerging selection strategy for improvement antibody targeting and drug delivery properties.
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Affiliation(s)
- Ana S. André
- Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, Lisbon, Portugal
- Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Lisbon, Portugal
| | - Isa Moutinho
- Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, Lisbon, Portugal
- Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Lisbon, Portugal
| | - Joana N. R. Dias
- Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, Lisbon, Portugal
- Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Lisbon, Portugal
| | - Frederico Aires-da-Silva
- Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, Lisbon, Portugal
- Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Lisbon, Portugal
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Jirapongpairoj W, Nozaki R, Koiwai K, Hirono I, Kondo H. Identification of a rabbit Ig light chain recombinant protein bound to serum immunoglobulins from different marine fish species. FISH & SHELLFISH IMMUNOLOGY 2022; 127:939-947. [PMID: 35868474 DOI: 10.1016/j.fsi.2022.07.032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 07/05/2022] [Accepted: 07/11/2022] [Indexed: 06/15/2023]
Abstract
The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column. The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig-specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig. The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library. A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting. The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting. The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins.
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Affiliation(s)
- Walissara Jirapongpairoj
- Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan
| | - Reiko Nozaki
- Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan
| | - Keiichiro Koiwai
- Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan
| | - Ikuo Hirono
- Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan
| | - Hidehiro Kondo
- Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan.
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Chen Z, Bao L, Zhu B, Fu H, Zhu S, Ji T, Xue Y, Liu C, Wang X, Li F, Lv Q, Qi F, Yu P, Deng W, Xu W, Qin C, Liu H, Jin Q. Structural and functional analysis of a potent human neutralizing antibody against enterovirus A71. SCIENCE CHINA LIFE SCIENCES 2022; 65:2517-2526. [PMID: 35696017 PMCID: PMC9189450 DOI: 10.1007/s11427-021-2095-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Accepted: 03/22/2022] [Indexed: 10/29/2022]
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39
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Duan Z, Buffington J, Hong J, Ho M. Production and Purification of Shark and Camel Single-Domain Antibodies from Bacterial and Mammalian Cell Expression Systems. Curr Protoc 2022; 2:e459. [PMID: 35714364 PMCID: PMC9219022 DOI: 10.1002/cpz1.459] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Single-domain antibodies, including the antigen-binding variable domains of the shark immunoglobulin new antigen receptor and the camelid variable region of the heavy chain, are the smallest antigen recognition domains (∼15 kDa) and have unique characteristics compared to conventional antibodies. They are capable of binding epitopes that are hard to access for classical antibodies and can also be used for therapeutics or diagnostics or as modular building blocks for multi-domain constructs, antibody-drug conjugates, immunotoxins, or chimeric antigen receptor therapy. This article contains detailed procedures for the purification and validation of two single-domain antibodies (one shark and one camel), which bind to the S2 subunit of the SARS-CoV-2 spike protein, using both bacterial and mammalian cell expression systems. It provides a comprehensive reference for the production of single-domain antibodies with high yield, good quality, and purity. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Production of single-domain antibodies from Escherichia coli Alternate Protocol: Production of single-domain antibodies using the mammalian cell line Expi293F Support Protocol 1: Production and purification of single-domain antibodies on a small scale with the polymyxin B method Support Protocol 2: Validation of single-domain antibodies by ELISA.
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Affiliation(s)
- Zhijian Duan
- Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Jesse Buffington
- Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Jessica Hong
- Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
| | - Mitchell Ho
- Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
- Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
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40
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Lin YS, Hsieh SJ, Tsai KC, Cheng MH, Yang TW, Lin TY, Chang FL, Chiang CW, Chen WC, Huang HT, Lee YC. Blockade effect of avian-derived anti-VISTA antibodies on immunosuppressive responses. ALL LIFE 2022. [DOI: 10.1080/26895293.2022.2063951] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Affiliation(s)
- Yun-Shih Lin
- Department of Psychiatry, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan
| | - Shang-Ju Hsieh
- Division of Urology, Department of Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan
| | - Keng-Chang Tsai
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Ming-Hui Cheng
- Department of Laboratory Medicine, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan, Taiwan
| | - Tz-Wen Yang
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
| | - Tsai-Yu Lin
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan
| | - Fu-Ling Chang
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan
| | - Chen-Wei Chiang
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Wang-Chuan Chen
- The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan
- Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan
| | - Hsien-Te Huang
- Department of Nursing, Meiho University, Pingtung, Taiwan
| | - Yu-Ching Lee
- Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
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41
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A novel and effective approach to generate germline-like monoclonal antibodies by integration of phage and mammalian cell display platforms. Acta Pharmacol Sin 2022; 43:954-962. [PMID: 34234269 PMCID: PMC8975860 DOI: 10.1038/s41401-021-00707-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Accepted: 05/25/2021] [Indexed: 12/13/2022]
Abstract
Phage display technology allows for rapid selection of antibodies from the large repertoire of human antibody fragments displayed on phages. However, antibody fragments should be converted to IgG for biological characterizations and affinity of antibodies obtained from phage display library is frequently not sufficient for efficient use in clinical settings. Here, we describe a new approach that combines phage and mammalian cell display, enabling simultaneous affinity screening of full-length IgG antibodies. Using this strategy, we successfully obtained a novel germline-like anti-TIM-3 monoclonal antibody named m101, which was revealed to be a potent anti-TIM-3 therapeutic monoclonal antibody via in vitro and in vivo experiments, indicating its effectiveness and power. Thus, this platform can help develop new monoclonal antibody therapeutics with high affinity and low immunogenicity.
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Michigami M, Ramanayake Mudiyanselage TMR, Suzuki M, Ishizako H, Notsu K, Sugiura K, Fujii I. New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix-Loop-Helix Peptide to Bind VEGF for Tumor Growth Inhibition. ACS Chem Biol 2022; 17:647-653. [PMID: 35176860 DOI: 10.1021/acschembio.1c00940] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
As a small affinity molecule to serve as an alternative to antibodies, we have developed a conformationally constrained peptide with a de novo designed helix-loop-helix (HLH) scaffold. To evaluate its potential for biomedical applications, we performed directed evolution of HLH peptides to obtain an inhibitor for vascular endothelial growth factor-A (VEGF). A phage-displayed library of HLH peptides was constructed and screened against VEGF, giving the peptide VS42 that inhibits the VEGF/VEGF receptor-2 interaction (IC50 = 210 nM), which was further improved by in vitro affinity maturation using a yeast-displayed library. An identified HLH peptide, VS42-LR3, exhibited improved inhibitory activity (IC50 = 37 nM), high thermal stability, and excellent resistance against chemical denaturation. In biological activity tests, the HLH peptide was found to block VEGF-induced proliferation of human umbilical vein endothelial cells and suppress tumor growth in a murine xenograft model of human colorectal cancer.
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Affiliation(s)
- Masataka Michigami
- Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Tharanga M. R. Ramanayake Mudiyanselage
- Department of Veterinary Science, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-58 Rinku-oraikita, Izumisano, Osaka 598-8531, Japan
| | - Miho Suzuki
- Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Hirotsugu Ishizako
- Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Kunpei Notsu
- Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Kikuya Sugiura
- Department of Veterinary Science, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-58 Rinku-oraikita, Izumisano, Osaka 598-8531, Japan
| | - Ikuo Fujii
- Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
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Dias JNR, Almeida A, André AS, Aguiar SI, Bule P, Nogueira S, Oliveira SS, Carrapiço B, Gil S, Tavares L, Aires-da-Silva F. Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy. Sci Rep 2022; 12:2678. [PMID: 35177658 PMCID: PMC8854400 DOI: 10.1038/s41598-022-06549-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Accepted: 01/20/2022] [Indexed: 12/02/2022] Open
Abstract
Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.
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Affiliation(s)
- Joana N R Dias
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - André Almeida
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Ana S André
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Sandra I Aguiar
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Pedro Bule
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Sara Nogueira
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Soraia S Oliveira
- Technophage SA, Avenida Prof. Egas Moniz, Edifício Egas Moniz, 1649-028, Lisbon, Portugal
| | - Belmira Carrapiço
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Solange Gil
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Luís Tavares
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal
| | - Frederico Aires-da-Silva
- CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, 1300-477, Lisbon, Portugal.
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Yue H, Li Y, Yang M, Mao C. T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2103645. [PMID: 34914854 PMCID: PMC8811829 DOI: 10.1002/advs.202103645] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Revised: 10/27/2021] [Indexed: 05/05/2023]
Abstract
Bacteriophages, also known as phages, are specific antagonists against bacteria. T7 phage has drawn massive attention in precision medicine owing to its distinctive advantages, such as short replication cycle, ease in displaying peptides and proteins, high stability and cloning efficiency, facile manipulation, and convenient storage. By introducing foreign gene into phage DNA, T7 phage can present foreign peptides or proteins site-specifically on its capsid, enabling it to become a nanoparticle that can be genetically engineered to screen and display a peptide or protein capable of recognizing a specific target with high affinity. This review critically introduces the biomedical use of T7 phage, ranging from the detection of serological biomarkers and bacterial pathogens, recognition of cells or tissues with high affinity, design of gene vectors or vaccines, to targeted therapy of different challenging diseases (e.g., bacterial infection, cancer, neurodegenerative disease, inflammatory disease, and foot-mouth disease). It also discusses perspectives and challenges in exploring T7 phage, including the understanding of its interactions with human body, assembly into scaffolds for tissue regeneration, integration with genome editing, and theranostic use in clinics. As a genetically modifiable biological nanoparticle, T7 phage holds promise as biomedical imaging probes, therapeutic agents, drug and gene carriers, and detection tools.
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Affiliation(s)
- Hui Yue
- School of Materials Science and EngineeringZhejiang UniversityHangzhouZhejiang310027P. R. China
| | - Yan Li
- Institute of Applied Bioresource ResearchCollege of Animal ScienceZhejiang UniversityYuhangtang Road 866HangzhouZhejiang310058P. R. China
| | - Mingying Yang
- Institute of Applied Bioresource ResearchCollege of Animal ScienceZhejiang UniversityYuhangtang Road 866HangzhouZhejiang310058P. R. China
| | - Chuanbin Mao
- School of Materials Science and EngineeringZhejiang UniversityHangzhouZhejiang310027P. R. China
- Department of Chemistry and BiochemistryStephenson Life Science Research CenterInstitute for Biomedical Engineering, Science and TechnologyUniversity of Oklahoma101 Stephenson ParkwayNormanOklahoma73019‐5251USA
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Avital-Shmilovici M, Liu X, Shaler T, Lowenthal A, Bourbon P, Snider J, Tambo-Ong A, Repellin C, Yniguez K, Sambucetti L, Madrid PB, Collins N. Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers. ACS CENTRAL SCIENCE 2022; 8:86-101. [PMID: 35106376 PMCID: PMC8796305 DOI: 10.1021/acscentsci.1c01041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Indexed: 06/14/2023]
Abstract
Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This platform utilizes the fiber-optic array scanning technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (∼83 000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic "self-readable" non-natural polymers that can be sequenced at the femtomole scale by chemical fragmentation and high-resolution mass spectrometry. The versatility and throughput of the platform were demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor 1 (ASGPR), IL-6, IL-6 receptor (IL-6R), and TNFα. Hits with low nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery of a nonglycan ligand.
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Chen Z, Zhang P, Matsuoka Y, Tsybovsky Y, West K, Santos C, Boyd LF, Nguyen H, Pomerenke A, Stephens T, Olia AS, De Giorgi V, Holbrook MR, Gross R, Postnikova E, Garza NL, Johnson RF, Margulies DH, Kwong PD, Alter HJ, Buchholz UJ, Lusso P, Farci P. Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2022. [PMID: 35043120 DOI: 10.1101/2022.01.12.22269023] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity 1 . This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies 1, 2 . Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe 3 . Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.
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Zhao Q, Chapman A, Huang Y, Ferguson M, McBride S, Kelly M, Weiner M, Li X. Ligand-Directed GPCR Antibody Discovery. Methods Mol Biol 2022; 2394:319-342. [PMID: 35094336 DOI: 10.1007/978-1-0716-1811-0_19] [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] [Indexed: 06/14/2023]
Abstract
Developing affinity reagents recognizing and modulating G-protein coupled receptors (GPCR) function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies exist on the market, but the success rate of development is still poor compared with antibodies targeting soluble or peripherally anchored proteins. More importantly, most of these antibodies do not modulate GPCR function. The current pipeline for antibody development primarily screens for overall affinity rather than functional epitope recognition. We developed a new strategy utilizing natural ligand affinity to generate a library of antibody variants with an inherent bias toward the active site of the GPCR. Instead of using phage libraries displaying antibodies with random CDR sequences at polymorphism sites observed in natural immune repertoire sequences, we generated focused antibody libraries with a natural ligand encoded within or conjugated to one of the CDRs or the N-terminus. To tailor antibody binding to the active site, we limited the sequence randomization of the antibody in regions holstering the ligand while leaving the ligand-carrying part unaltered in the first round of randomization. With hits from the successful first round, the second round of randomization of the ligand-carrying part was then performed to eliminate the bias of the ligand. Based on our results on three different GPCR targets, the proposed pipeline will enable the rapid generation of functional antibodies (both agonists and antagonists) against high-value targets with poor function epitope exposures including GPCR, channels, transporters as well as cell surface targets whose binding site is heavily masked by glycosylation.
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Affiliation(s)
- Qi Zhao
- Abcam plc, Branford, CT, USA.
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Ferrara F, D’Angelo S, Erasmus MF, Teixeira AA, Leal-Lopes C, Spector LP, Pohl T, Fanni A, Cocklin S, Bradbury ARM. Pandemic's silver lining. MAbs 2022; 14:2133666. [PMID: 36253351 PMCID: PMC9578449 DOI: 10.1080/19420862.2022.2133666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/09/2023] Open
Abstract
The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.
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Affiliation(s)
| | - Sara D’Angelo
- Specifica Inc., A Q2 Solutions Company, Santa Fe, NM, USA
| | | | | | | | | | - Tom Pohl
- Specifica Inc., A Q2 Solutions Company, Santa Fe, NM, USA
| | - Adeline Fanni
- Specifica Inc., A Q2 Solutions Company, Santa Fe, NM, USA
| | - Simon Cocklin
- Specifica Inc., A Q2 Solutions Company, Santa Fe, NM, USA
| | - Andrew R. M. Bradbury
- Specifica Inc., A Q2 Solutions Company, Santa Fe, NM, USA,CONTACT Andrew R. M. Bradbury Specifica Inc, Los Alamos, NM, USA
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Schneider KT, Kirmann T, Wenzel EV, Grosch JH, Polten S, Meier D, Becker M, Matejtschuk P, Hust M, Russo G, Dübel S. Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization. Front Cell Infect Microbiol 2021; 11:717689. [PMID: 34869052 PMCID: PMC8634725 DOI: 10.3389/fcimb.2021.717689] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2021] [Accepted: 10/25/2021] [Indexed: 11/18/2022] Open
Abstract
Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies' initial properties after storage, without any drop in affinity for any of the tested antibody clones.
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Affiliation(s)
- Kai-Thomas Schneider
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Toni Kirmann
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Esther Veronika Wenzel
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
- Abcalis GmbH, Braunschweig, Germany
| | - Jan-Hendrik Grosch
- Institute of Biochemical Engineering, Technische Universität Braunschweig, Braunschweig, Germany
- Center of Pharmaceutical Engineering, Technische Universität Braunschweig, Braunschweig, Germany
| | - Saskia Polten
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Doris Meier
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Marlies Becker
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Paul Matejtschuk
- Standardisation Science, National Institute for Biological Standards & Control (NIBSC), Hertfordshire, United Kingdom
| | - Michael Hust
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Giulio Russo
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
- Abcalis GmbH, Braunschweig, Germany
| | - Stefan Dübel
- Department of Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
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50
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Jaroszewicz W, Morcinek-Orłowska J, Pierzynowska K, Gaffke L, Węgrzyn G. Phage display and other peptide display technologies. FEMS Microbiol Rev 2021; 46:6407522. [PMID: 34673942 DOI: 10.1093/femsre/fuab052] [Citation(s) in RCA: 141] [Impact Index Per Article: 35.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Accepted: 10/19/2021] [Indexed: 12/13/2022] Open
Abstract
Phage display technology, which is based on the presentation of peptide sequences on the surface of bacteriophage virions, was developed over 30 years ago. Improvements in phage display systems have allowed us to employ this method in numerous fields of biotechnology, as diverse as immunological and biomedical applications, the formation of novel materials and many others. The importance of phage display platforms was recognized by awarding the Nobel Prize in 2018 "for the phage display of peptides and antibodies". In contrast to many review articles concerning specific applications of phage display systems published in recent years, we present an overview of this technology, including a comparison of various display systems, their advantages and disadvantages, and examples of applications in various fields of science, medicine, and the broad sense of biotechnology. Other peptide display technologies, which employ bacterial, yeast and mammalian cells, as well as eukaryotic viruses and cell-free systems, are also discussed. These powerful methods are still being developed and improved; thus, novel sophisticated tools based on phage display and other peptide display systems are constantly emerging, and new opportunities to solve various scientific, medical and technological problems can be expected to become available in the near future.
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Affiliation(s)
- Weronika Jaroszewicz
- Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
| | | | - Karolina Pierzynowska
- Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
| | - Lidia Gaffke
- Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
| | - Grzegorz Węgrzyn
- Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
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