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1
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Wang M, Zhang Z, Yang Y, Peng X, Yin H. A targeted MAVS fusion protein for controlled innate immune activation and antitumor therapy. Oncoimmunology 2025; 14:2478850. [PMID: 40085508 PMCID: PMC11913393 DOI: 10.1080/2162402x.2025.2478850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2025] [Revised: 02/25/2025] [Accepted: 03/06/2025] [Indexed: 03/16/2025] Open
Abstract
Targeted therapies leveraging the innate immune system are emerging as promising cancer treatments. The mitochondrial antiviral signaling protein (MAVS) plays a crucial role in initiating innate immune responses, but its clinical use is limited by the risk of uncontrolled activation and systemic toxicity. To address this, we developed a novel therapeutic agent, the truncated interferon activation switch (TRIAS), combining MAVS truncates with a tumor antigen-targeting single-chain variable fragment (scFv). This design ensures antigen-dependent, controlled activation. Lentiviral delivery of TRIAS induced significant antitumor responses, including complete tumor regression in some cases. Flow cytometry (FCM) analysis further confirmed that tumor cells were the predominant population expressing the transgene. TRIAS-expressing tumor cells exhibited enhanced antitumor activity, likely due to increased cytokine release and upregulated major histocompatibility complex (MHC) expression, enabling tumor cells to function as antigen-presenting cells. This activated other immune cells, driving adaptive immune responses. Additionally, TRIAS promoted a proinflammatory shift in the tumor microenvironment (TME). In conclusion, TRIAS was validated as an innovative immunotherapeutic agent with MAVS-like immune-activating properties and tightly controlled mechanisms, offering a safer and more effective approach for clinical cancer immunotherapy.
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Affiliation(s)
- Muhan Wang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
| | - Zhijie Zhang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
| | - YouYou Yang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
| | - Xiaoyi Peng
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
| | - Hongping Yin
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
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2
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Carbone C, De Luca R, Puca E, Agostini A, Caggiano A, Priori L, Esposito A, Ascrizzi S, Piro G, Salvatore L, De Sanctis F, Ugel S, Corbo V, Neri D, Tortora G. Antibody-based delivery of interleukin-2 modulates the immunosuppressive tumor microenvironment and achieves cure in pancreatic ductal adenocarcinoma syngeneic mice. J Exp Clin Cancer Res 2025; 44:7. [PMID: 39773310 PMCID: PMC11705946 DOI: 10.1186/s13046-024-03238-x] [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: 09/30/2024] [Accepted: 11/24/2024] [Indexed: 01/11/2025] Open
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly type of cancer, with an extremely low five-year overall survival rate. To date, current treatment options primarily involve various chemotherapies, which often prove ineffective and are associated with substantial toxicity. Furthermore, immunotherapies utilizing checkpoint inhibitors have shown limited efficacy in this context, highlighting an urgent need for novel therapeutic strategies. This study investigates the preclinical efficacy of an innovative targeted therapy based on antibody-cytokine fusion proteins, specifically interleukin-2 (IL-2), a pivotal driver of cell-mediated immunity, fused to L19 antibody, which selectively binds to extra domain B of fibronectin (EDB-FN1) expressed in the tumor microenvironment. METHODS We tested the effectiveness of different immunocytokines through in vivo characterization in syngeneic C57BL/6J orthotopic mouse models of PDAC. Based on these results, we decided to focus on L19-IL2. To assess the efficacy of this immunocytokine we developed an ex-vivo immune-spheroid interaction platform derived from murine 3D pancreatic cultures, and telomerase reverse transcriptase (TERT) specific T-lymphocytes. Moreover, we evaluated the anti-cancer effect of L19-IL2 in combination with standard therapy in vivo experiments in PDAC mouse models. Tumor samples collected after the treatments were characterized for tumor infiltrating immune cell components by bulk RNA sequencing (RNA-seq) and spatial transcriptomics (Stereo-seq) analysis. RESULTS The tumor-targeted L19-IL2 fusion protein demonstrated potent, dose-dependent anti-tumor activity in mice with pancreatic tumors resistant to standard chemotherapy. Spatial Transcriptomics (ST) and RNA-seq analyses indicated that L19-IL2 treatment induced a significant influx of immune cells into the tumor microenvironment, with these cells expressing activation markers like granzymes, perforins, and the IL-2 receptors. CONCLUSIONS Our results demonstrated that L19-IL2 enhances immune infiltration and cytotoxicity, remodeling the "cold" tumor microenvironment (TME) in PDAC. This innovative antibody-cytokine fusion protein improves therapeutic outcomes, paving the way for novel targeted treatment strategies in PDAC.
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Affiliation(s)
- Carmine Carbone
- Department of Medical and Surgical Sciences, Medical Oncology , Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy
| | - Roberto De Luca
- Philochem AG, Libernstrasse 3, Otelfingen, 8112, Switzerland
| | - Emanuele Puca
- Philochem AG, Libernstrasse 3, Otelfingen, 8112, Switzerland
| | - Antonio Agostini
- Department of Medical and Surgical Sciences, Medical Oncology , Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy
- Bioinformatics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy
| | - Alessia Caggiano
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy
| | - Lorenzo Priori
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy
| | - Annachiara Esposito
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy
| | - Serena Ascrizzi
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy
| | - Geny Piro
- Department of Medical and Surgical Sciences, Medical Oncology , Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy
| | - Lisa Salvatore
- Department of Medical and Surgical Sciences, Medical Oncology , Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy
| | - Francesco De Sanctis
- Department of Medicine, Section of Immunology, University of Verona, Verona, Italy
| | - Stefano Ugel
- Department of Medicine, Section of Immunology, University of Verona, Verona, Italy
| | - Vincenzo Corbo
- Department of Diagnostics and Public Health, University of Verona, Verona, Italy
- ARC-Net Research Centre, University of Verona, Verona, Italy
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich, CH-8093, Switzerland
- Philogen Spa, Piazza La Lizza 7, Siena, 53100, Italy
| | - Giampaolo Tortora
- Department of Medical and Surgical Sciences, Medical Oncology , Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS, Rome, Italy.
- Department of Translational Medicine, Medical Oncology , Catholic University of the Sacred Heart, Rome, Italy.
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3
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Prodi E, Corbellari R, Ghezzi L, Ciambellotti S, Catastini JE, Rappuoli M, Rotta G, Sakic I, Weiss T, Weller M, Pellegrino C, Manz MG, Neri D, De Luca R. Tripokin: A multi-specific immunocytokine for cancer immunotherapy. Int J Cancer 2025; 156:216-228. [PMID: 39177452 DOI: 10.1002/ijc.35145] [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: 03/12/2024] [Revised: 07/10/2024] [Accepted: 07/31/2024] [Indexed: 08/24/2024]
Abstract
Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 μg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites.
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Affiliation(s)
- Eleonora Prodi
- Philochem AG, Otelfingen, Switzerland
- CiBIO (Department of Cellular, Computational and Integrative Biology), University of Trento, Trento, Italy
| | - Riccardo Corbellari
- Philochem AG, Otelfingen, Switzerland
- CiBIO (Department of Cellular, Computational and Integrative Biology), University of Trento, Trento, Italy
| | | | | | | | | | | | - Irma Sakic
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Tobias Weiss
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Michael Weller
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Christian Pellegrino
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Markus G Manz
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland
| | - Dario Neri
- Philogen Spa, Siena, Italy
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zurich, Switzerland
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4
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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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5
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Di Nitto C, Ravazza D, Gilardoni E, Look T, Sun M, Prodi E, Moisoiu V, Pellegrino C, Manz MG, Puca E, Weller M, Weiss T, Neri D, De Luca R. An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade. J Immunother Cancer 2024; 12:e008504. [PMID: 39142716 PMCID: PMC11332014 DOI: 10.1136/jitc-2023-008504] [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] [Accepted: 07/27/2024] [Indexed: 08/16/2024] Open
Abstract
BACKGROUND Anti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable combination partners to predict or further improve the activity of immune checkpoint inhibitors. Antibody-cytokine fusions are a class of pharmaceuticals that showed the potential to boost the anticancer properties of other immunotherapies. Extradomain A-fibronectin (EDA-FN), which is expressed in most solid and hematological tumors but is virtually undetectable in healthy adult tissues, is an attractive target for the delivery of cytokine at the site of the disease. METHODS In this work, we describe the generation and characterization of a novel interleukin-7-based fusion protein targeting EDA-FN termed F8(scDb)-IL7. The product consists of the F8 antibody specific to the alternatively spliced EDA of FN in the single-chain diabody (scDb) format fused to human IL-7. RESULTS F8(scDb)-IL7 efficiently stimulates human peripheral blood mononuclear cells in vitro. Moreover, the product significantly increases the expression of T Cell Factor 1 (TCF-1) on CD8+T cells compared with an IL2-fusion protein. TCF-1 has emerged as a pivotal transcription factor that influences the durability and potency of immune responses against tumors. In preclinical cancer models, F8(scDb)-IL7 demonstrates potent single-agent activity and eradicates sarcoma lesions when combined with anti-PD-1. CONCLUSIONS Our results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.
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Affiliation(s)
| | | | | | - Thomas Look
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | - Miaomiao Sun
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | | | - Vlad Moisoiu
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | - Christian Pellegrino
- Department of Medical Oncology and Hematology, University Hospital Zurich, Zurich, Switzerland
| | - Markus G. Manz
- Department of Medical Oncology and Hematology, University Hospital Zurich, Zurich, Switzerland
| | | | - Michael Weller
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
- University of Zurich, Zurich, Switzerland
| | - Tobias Weiss
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
- University of Zurich, Zurich, Switzerland
| | - Dario Neri
- Philogen SpA, Siena, Italy
- Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
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6
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Ribeiro R, Moreira JN, Goncalves J. Development of a new affinity maturation protocol for the construction of an internalizing anti-nucleolin antibody library. Sci Rep 2024; 14:10608. [PMID: 38719911 PMCID: PMC11079059 DOI: 10.1038/s41598-024-61230-z] [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: 10/13/2023] [Accepted: 05/02/2024] [Indexed: 05/12/2024] Open
Abstract
Over the last decades, monoclonal antibodies have substantially improved the treatment of several conditions. The continuous search for novel therapeutic targets and improvements in antibody's structure, demands for a constant optimization of their development. In this regard, modulation of an antibody's affinity to its target has been largely explored and culminated in the discovery and optimization of a variety of molecules. It involves the creation of antibody libraries and selection against the target of interest. In this work, we aimed at developing a novel protocol to be used for the affinity maturation of an antibody previously developed by our group. An antibody library was constructed using an in vivo random mutagenesis approach that, to our knowledge, has not been used before for antibody development. Then, a cell-based phage display selection protocol was designed to allow the fast and simple screening of antibody clones capable of being internalized by target cells. Next generation sequencing coupled with computer analysis provided an extensive characterization of the created library and post-selection pool, that can be used as a guide for future antibody development. With a single selection step, an enrichment in the mutated antibody library, given by a decrease in almost 50% in sequence diversity, was achieved, and structural information useful in the study of the antibody-target interaction in the future was obtained.
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Affiliation(s)
- Rita Ribeiro
- CNC-Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), Faculty of Medicine (Polo 1), University of Coimbra, Coimbra, Portugal
- Faculty of Pharmacy, iMed.ULisboa - Research Institute for Medicines, University of Lisbon, Lisbon, Portugal
- Univ Coimbra-University of Coimbra, CIBB, Faculty of Pharmacy, Coimbra, Portugal
| | - João N Moreira
- CNC-Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), Faculty of Medicine (Polo 1), University of Coimbra, Coimbra, Portugal.
- Univ Coimbra-University of Coimbra, CIBB, Faculty of Pharmacy, Coimbra, Portugal.
| | - João Goncalves
- Faculty of Pharmacy, iMed.ULisboa - Research Institute for Medicines, University of Lisbon, Lisbon, Portugal.
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7
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Shaw TI, Wagner J, Tian L, Wickman E, Poudel S, Wang J, Paul R, Koo SC, Lu M, Sheppard H, Fan Y, O'Neill FH, Lau CC, Zhou X, Zhang J, Gottschalk S. Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression. Nat Commun 2024; 15:3732. [PMID: 38702309 PMCID: PMC11068777 DOI: 10.1038/s41467-024-47649-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Accepted: 04/09/2024] [Indexed: 05/06/2024] Open
Abstract
Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.
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Affiliation(s)
- Timothy I Shaw
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
- Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA
| | - Jessica Wagner
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Liqing Tian
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Elizabeth Wickman
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
- Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Suresh Poudel
- Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Jian Wang
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Robin Paul
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Selene C Koo
- Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Meifen Lu
- Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Heather Sheppard
- Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Yiping Fan
- Center for Applied Bioinformatics, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Francis H O'Neill
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
| | - Ching C Lau
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
- Connecticut Children's Medical Center, Hartford, CT, 06106, USA
- University of Connecticut School of Medicine, Farmington, CT, 06032, USA
| | - Xin Zhou
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Jinghui Zhang
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.
| | - Stephen Gottschalk
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.
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8
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Pekar L, Krah S, Zielonka S. Taming the beast: engineering strategies and biomedical potential of antibody-based cytokine mimetics. Expert Opin Biol Ther 2024:1-4. [PMID: 38385844 DOI: 10.1080/14712598.2024.2322062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Accepted: 02/15/2024] [Indexed: 02/23/2024]
Affiliation(s)
- Lukas Pekar
- Antibody Discovery & Protein Engineering, Merck Healthcare KGaA, Darmstadt, Germany
| | - Simon Krah
- Antibody Discovery & Protein Engineering, Merck Healthcare KGaA, Darmstadt, Germany
| | - Stefan Zielonka
- Antibody Discovery & Protein Engineering, Merck Healthcare KGaA, Darmstadt, Germany
- Biomolecular Immunotherapy, Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany
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9
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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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10
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Ferrara F, Fanni A, Teixeira AAR, Molina E, Leal-Lopes C, DeAguero A, D'Angelo S, Erasmus MF, Spector L, Rodriguez Carnero LA, Li J, Pohl TJ, Suslov N, Desrumeaux K, McMahon C, Kathuria S, Bradbury ARM. A next-generation Fab library platform directly yielding drug-like antibodies with high affinity, diversity, and developability. MAbs 2024; 16:2394230. [PMID: 39192463 DOI: 10.1080/19420862.2024.2394230] [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: 06/20/2024] [Revised: 08/13/2024] [Accepted: 08/15/2024] [Indexed: 08/29/2024] Open
Abstract
We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.
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Affiliation(s)
| | - Adeline Fanni
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
| | | | - Esteban Molina
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
| | | | | | - Sara D'Angelo
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
| | | | - Laura Spector
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
| | | | - Jianquan Li
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
| | - Thomas J Pohl
- Specifica LLC, a Q2 Solutions Company, Santa Fe, NM, USA
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11
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Marchetti A, Lima WC, Hammel P, Cosson P. A quantitative comparison of antibodies against epitope tags for immunofluorescence detection. FEBS Open Bio 2023; 13:2239-2245. [PMID: 37702273 PMCID: PMC10699100 DOI: 10.1002/2211-5463.13705] [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: 05/30/2023] [Revised: 07/21/2023] [Accepted: 09/11/2023] [Indexed: 09/14/2023] Open
Abstract
Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side-by-side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti-IL2Ra antibody and the signal generated by the anti-epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: 'good' antibodies generated high signals even when used at a low concentration (50 ng·mL-1 ), 'fair' antibodies generated a high signal only at high concentrations (5000 ng·mL-1 ), and 'mediocre' antibodies generated positive but weak signals. Except for an anti-myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side-by-side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies.
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Affiliation(s)
- Anna Marchetti
- Department of Cell Physiology and Metabolism, Faculty of MedicineUniversity of GenevaSwitzerland
| | - Wanessa C. Lima
- Department of Cell Physiology and Metabolism, Faculty of MedicineUniversity of GenevaSwitzerland
| | - Philippe Hammel
- Department of Cell Physiology and Metabolism, Faculty of MedicineUniversity of GenevaSwitzerland
| | - Pierre Cosson
- Department of Cell Physiology and Metabolism, Faculty of MedicineUniversity of GenevaSwitzerland
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12
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Tang J, Liu N, Zhu Y, Li Y, Zhao X. CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells. Immunol Invest 2023; 52:985-996. [PMID: 37815216 DOI: 10.1080/08820139.2023.2264332] [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: 10/11/2023]
Abstract
BACKGROUND CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. METHODS We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. RESULTS In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells' normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. CONCLUSION APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.
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Affiliation(s)
- Jie Tang
- Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and Department of Respiratory and Critical care Medicine and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Nan Liu
- Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and Department of Respiratory and Critical care Medicine and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yongjie Zhu
- Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and Department of Respiratory and Critical care Medicine and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yi Li
- Core Facilities, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China
| | - Xudong Zhao
- Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and Department of Respiratory and Critical care Medicine and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China
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13
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Feng Y, Hao Y, Wang Y, Song W, Zhang S, Ni D, Yan F, Sun L. Ultrasound Molecular Imaging of Bladder Cancer via Extradomain B Fibronectin-Targeted Biosynthetic GVs. Int J Nanomedicine 2023; 18:4871-4884. [PMID: 37662687 PMCID: PMC10474871 DOI: 10.2147/ijn.s412422] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2023] [Accepted: 08/11/2023] [Indexed: 09/05/2023] Open
Abstract
Purpose Ultrasound molecular imaging (UMI) has proven promising to diagnose the onset and progression of diseases such as angiogenesis, inflammation, and thrombosis. However, microbubble-based acoustic probes are confined to intravascular targets due to their relatively large particle size, greatly reducing the application value of UMI, especially for extravascular targets. Extradomain B fibronectin (ED-B FN) is an important glycoprotein associated with tumor genesis and development and highly expressed in many types of tumors. Here, we developed a gas vesicles (GVs)-based nanoscale acoustic probe (ZD2-GVs) through conjugating ZD2 peptides which can specially target to ED-B FN to the biosynthetic GVs. Materials and Methods ED-B FN expression was evaluated in normal liver and tumor tissues with immunofluorescence and Western blot. ZD2-GVs were prepared by conjugating ZD2 to the surface of GVs by amide reaction. The inverted microscope was used to analyze the targeted binding capacity of ZD2-GVs to MB49 cells (bladder cancer cell line). The contrast-enhanced imaging features of GVs, non-targeted control GVs (CTR-GVs), and targeted GVs (ZD2-GVs) were compared in three MB49 tumor mice. The penetration ability of ZD2-GVs in tumor tissues was assessed by fluorescence immunohistochemistry. The biosafety of GVs was evaluated by CCK8, blood biochemistry, and HE staining. Results Strong ED-B FN expression was observed in tumor tissues while little expression in normal liver tissues. The resulting ZD2-GVs had only 267.73 ± 2.86 nm particle size and exhibited excellent binding capability to the MB49 tumor cells. The in vivo UMI experiments showed that ZD2-GVs produced stronger and longer retention in the BC tumors than that of the non-targeted CTR-GVs and GVs. Fluorescence immunohistochemistry confirmed that ZD2-GVs could penetrate the tumor vascular into the interstitial space of the tumors. Biosafety analysis revealed there was no significant cytotoxicity to these tested mice. Conclusion Thus, ZD2-GVs can function as a potential UMI probe for the early diagnosis of bladder cancer.
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Affiliation(s)
- Yanan Feng
- Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, People’s Republic of China
- Department of Abdominal Ultrasound, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266000, People’s Republic of China
| | - Yongsheng Hao
- Center for Cell and Gene Circuit Design, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People’s Republic of China
| | - Yuanyuan Wang
- Center for Cell and Gene Circuit Design, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People’s Republic of China
| | - Weijian Song
- Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, People’s Republic of China
- Bengbu Medical College, Bengbu, 233030, People's Republic of China
| | - Shanxin Zhang
- Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, People’s Republic of China
| | - Dong Ni
- Medical Ultrasound Image Computing (MUSIC) Laboratory, Shenzhen University, Shenzhen, 518055, People’s Republic of China
| | - Fei Yan
- Center for Cell and Gene Circuit Design, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People’s Republic of China
| | - Litao Sun
- Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, People’s Republic of China
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14
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Zhang Z, Liu C, Wang M, Sun R, Yang Z, Hua Z, Wu Y, Wu M, Wang H, Qiu W, Yin H, Yang M. Treating solid tumors with TCR-based chimeric antigen receptor targeting extra domain B-containing fibronectin. J Immunother Cancer 2023; 11:e007199. [PMID: 37586774 PMCID: PMC10432677 DOI: 10.1136/jitc-2023-007199] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/27/2023] [Indexed: 08/18/2023] Open
Abstract
BACKGROUND The suppression of chimeric antigen receptor (CAR) T cells by the tumor microenvironment (TME) is a crucial obstacle in the T-cell-based treatment of solid tumors. Extra domain B (EDB)-fibronectin is an oncofetal antigen expressed on the endothelium layer of the neovasculature and cancer cells. Though recognized as a T cell therapy target, engineered CAR T cells thus far have failed to demonstrate satisfactory in vivo efficacy. In this study, we report that targeting EDB-fibronectin by redirected TCR-CAR T cells (rTCR-CAR) bypasses the suppressive TME for solid tumor treatment and sufficiently suppressed tumor growth.We generated EDB-targeting CAR by fusing single-chain variable fragment to CD3ε, resulting in rTCR-CAR. Human primary T cells and Jurkat cells were used to study the EDB-targeting T cells. Differences to the traditional second-generation CAR T cell in signaling, immune synapse formation, and T cell exhaustion were characterized. Cytotoxicity of the rTCR-CAR T cells was tested in vitro, and therapeutic efficacies were demonstrated using xenograft models. METHODS RESULTS: In the xenograft models, the rTCR-CAR T cells demonstrated in vivo efficacies superior to that based on traditional CAR design. A significant reduction in tumor vessel density was observed alongside tumor growth inhibition, extending even to tumor models established with EDB-negative cancer cells. The rTCR-CAR bound to immobilized EDB, and the binding led to immune synapse structures superior to that formed by second-generation CARs. By a mechanism similar to that for the conventional TCR complex, EDB-fibronectin activated the rTCR-CAR, resulting in rTCR-CAR T cells with low basal activation levels and increased in vivo expansion. CONCLUSION Our study has demonstrated the potential of rTCR-CAR T cells targeting the EDB-fibronectin as an anticancer therapeutic. Engineered to possess antiangiogenic and cytotoxic activities, the rTCR-CAR T cells showed therapeutic efficacies not impacted by the suppressive TMEs. These combined characteristics of a single therapeutic agent point to its potential to achieve sustained control of solid tumors.
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Affiliation(s)
- Zhijie Zhang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Chang Liu
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Muhan Wang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Rongcheng Sun
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
- Jiangsu Cell Tech Medical Research Institute, Nanjing, Jiangsu, China
| | - Zhe Yang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Zhen Hua
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Yushuang Wu
- Jiangsu Cell Tech Medical Research Institute, Nanjing, Jiangsu, China
| | - Mengting Wu
- Jiangsu Cell Tech Medical Research Institute, Nanjing, Jiangsu, China
| | - Hang Wang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Wen Qiu
- Department of Immunology, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Hongping Yin
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China
| | - Meijia Yang
- Jiangsu Cell Tech Medical Research Institute, Nanjing, Jiangsu, China
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Guo S, Zheng Y, Gao Z, Duan M, Liu S, Du P, Xu X, Xu K, Zhao X, Chai Y, Wang P, Zhao Q, Gao GF, Dai L. Dosing interval regimen shapes potency and breadth of antibody repertoire after vaccination of SARS-CoV-2 RBD protein subunit vaccine. Cell Discov 2023; 9:79. [PMID: 37507370 PMCID: PMC10382582 DOI: 10.1038/s41421-023-00585-5] [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: 03/14/2023] [Accepted: 07/09/2023] [Indexed: 07/30/2023] Open
Abstract
Vaccination with different vaccines has been implemented globally to counter the continuous COVID-19 pandemic. However, the vaccine-elicited antibodies have reduced efficiency against the highly mutated Omicron sub-variants. Previously, we developed a protein subunit COVID-19 vaccine called ZF2001, based on the dimeric receptor-binding domain (RBD). This vaccine has been administered using different dosing intervals in real-world setting. Some individuals received three doses of ZF2001, with a one-month interval between each dose, due to urgent clinical requirements. Others had an extended dosing interval of up to five months between the second and third dose, a standard vaccination regimen for the protein subunit vaccine against hepatitis B. In this study, we profile B cell responses in individuals who received three doses of ZF2001, and compared those with long or short dosing intervals. We observed that the long-interval group exhibited higher and broader serologic antibody responses. These responses were associated with the increased size and evolution of vaccine-elicited B-cell receptor repertoires, characterized by the elevation of expanded clonotypes and somatic hypermutations. Both groups of individuals generated substantial amounts of broadly neutralizing antibodies (bnAbs) against various SARS-CoV-2 variants, including Omicron sub-variants such as XBB. These bnAbs target four antigenic sites within the RBD. To determine the vulnerable site of SARS-CoV-2, we employed cryo-electron microscopy to identify the epitopes of highly potent bnAbs that targeted two major sites. Our findings provide immunological insights into the B cell responses elicited by RBD-based vaccine, and suggest that a vaccination regimen of prolonging time interval should be used in practice.
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Affiliation(s)
- Shuxin Guo
- Faculty of Health Sciences, University of Macau, Macau SAR, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China
| | - Yuxuan Zheng
- CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| | - Zhengrong Gao
- Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, China
- Shenzhen Children's Hospital, Shenzhen, Guangdong, China
| | - Minrun Duan
- School of Life Sciences, Yunnan University, Kunming, Yunnan, China
| | - Sheng Liu
- Department of Biology, Cryo-EM Center, Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Pan Du
- Vazyme Biotech, Nanjing, Jiangsu, China
| | - XiaoYu Xu
- Vazyme Biotech, Nanjing, Jiangsu, China
| | - Kun Xu
- Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
| | - Xin Zhao
- CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| | - Yan Chai
- CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| | - Peiyi Wang
- Department of Biology, Cryo-EM Center, Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Qi Zhao
- MoE Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - George F Gao
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China.
- CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
- Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China.
| | - Lianpan Dai
- CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
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16
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Bauer A, Klassa S, Herbst A, Maccioni C, Abhamon W, Segueni N, Kaluzhny Y, Hunter MC, Halin C. Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery. Pharmaceutics 2023; 15:1841. [PMID: 37514028 PMCID: PMC10385607 DOI: 10.3390/pharmaceutics15071841] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Revised: 06/09/2023] [Accepted: 06/21/2023] [Indexed: 07/30/2023] Open
Abstract
Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM's T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM-CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM-ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.
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Affiliation(s)
- Aline Bauer
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - Sven Klassa
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - Anja Herbst
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - Cristina Maccioni
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - William Abhamon
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - Noria Segueni
- Artimmune SAS, 13 Avenue Buffon, 45100 Orleans, France
| | - Yulia Kaluzhny
- MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA
| | - Morgan Campbell Hunter
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
| | - Cornelia Halin
- Institute of Pharmaceutical Sciences, ETH Zurich, 1-5/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland
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17
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Look T, Puca E, Bühler M, Kirschenbaum D, De Luca R, Stucchi R, Ravazza D, Di Nitto C, Roth P, Katzenelenbogen Y, Weiner A, Rindlisbacher L, Becher B, Amit I, Weller M, Neri D, Hemmerle T, Weiss T. Targeted delivery of tumor necrosis factor in combination with CCNU induces a T cell-dependent regression of glioblastoma. Sci Transl Med 2023; 15:eadf2281. [PMID: 37224228 DOI: 10.1126/scitranslmed.adf2281] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Accepted: 05/01/2023] [Indexed: 05/26/2023]
Abstract
Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192).
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Affiliation(s)
- Thomas Look
- Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich 8091, Switzerland
| | | | - Marcel Bühler
- Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich 8091, Switzerland
| | - Daniel Kirschenbaum
- Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
| | | | | | | | | | - Patrick Roth
- Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich 8091, Switzerland
| | | | - Assaf Weiner
- Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Lukas Rindlisbacher
- Institute of Experimental Immunology, University of Zurich, Zurich 8057, Switzerland
| | - Burkhard Becher
- Institute of Experimental Immunology, University of Zurich, Zurich 8057, Switzerland
| | - Ido Amit
- Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Michael Weller
- Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich 8091, Switzerland
| | | | | | - Tobias Weiss
- Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich 8091, Switzerland
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Abstract
As a natural function, antibodies defend the host from infected cells and pathogens by recognizing their pathogenic determinants. Antibodies (Abs) gained wide acceptance with an enormous impact on human health and have predominantly captured the arena of bio-therapeutics and bio-diagnostics. The scope of Ab-based biologics is vast, and it is likely to solve many unmet clinical needs in future. The majority of attention is now devoted to developing innovative technologies for manufacturing and engineering Abs, better suited to satisfy human needs. The advent of Ab engineering technologies (AET) led to phenomenal developments leading to the generation of Abs-/Ab-derived molecules with desirable functional properties proportional to their expanding requirements. Evolution brought by AET, from the naturally occurring Ab forms to several advanced Ab formats and derivatives, was much needed as it is of great interest to the pharmaceutical industry. Thus, numerous advancements in AET have propelled success in therapeutic Ab development, along with the potential for ever-increasing improvements. Unique characteristics of Abs, such as its diversity, specificity, structural integrity and an array of possible applications, together inspire continuous innovation in the field. Overall, the AET could assist in conquer of several limitations of Abs in terms of their applicability in the field of therapeutics, diagnostics and research; AET has so far led to the production of next-generation Abs, which have revolutionized these arenas. Here in this review, we discuss the various distinguished engineering platforms for Ab development and the progress in modern therapeutics by the so-called "next-generation Abs."
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Affiliation(s)
- Divya Kandari
- Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
| | - Rakesh Bhatnagar
- Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.,Banaras Hindu University, Varanasi, India.,Amity University Rajasthan, Jaipur, India
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Antitumor Therapy Targeting the Tumor Microenvironment. JOURNAL OF ONCOLOGY 2023; 2023:6886135. [PMID: 36908706 PMCID: PMC10005879 DOI: 10.1155/2023/6886135] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Revised: 02/13/2023] [Accepted: 02/20/2023] [Indexed: 03/06/2023]
Abstract
The development and progression of tumors in human tissues extensively rely on its surrounding environment, that is, tumor microenvironment which includes a variety of cells, molecules, and blood vessels. These components are modified, organized, and integrated to support and facilitate the growth, invasion, and metabolism of tumor cells, suggesting them as potential therapeutic targets in anticancer treatment. An increasing number of pharmacological agents have been developed and clinically applied to target the oncogenic components in the tumor microenvironment, and in this review, we will summarize these pharmacological agents that directly or indirectly target the cellular or molecular components in the tumor microenvironment. However, difficulties and challenges still exist in this field, which will also be reported in this literature.
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20
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Faisal SM, Comba A, Varela ML, Argento AE, Brumley E, Abel C, Castro MG, Lowenstein PR. The complex interactions between the cellular and non-cellular components of the brain tumor microenvironmental landscape and their therapeutic implications. Front Oncol 2022; 12:1005069. [PMID: 36276147 PMCID: PMC9583158 DOI: 10.3389/fonc.2022.1005069] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Accepted: 09/20/2022] [Indexed: 11/26/2022] Open
Abstract
Glioblastoma (GBM), an aggressive high-grade glial tumor, is resistant to therapy and has a poor prognosis due to its universal recurrence rate. GBM cells interact with the non-cellular components in the tumor microenvironment (TME), facilitating their rapid growth, evolution, and invasion into the normal brain. Herein we discuss the complexity of the interactions between the cellular and non-cellular components of the TME and advances in the field as a whole. While the stroma of non-central nervous system (CNS) tissues is abundant in fibrillary collagens, laminins, and fibronectin, the normal brain extracellular matrix (ECM) predominantly includes proteoglycans, glycoproteins, and glycosaminoglycans, with fibrillary components typically found only in association with the vasculature. However, recent studies have found that in GBMs, the microenvironment evolves into a more complex array of components, with upregulated collagen gene expression and aligned fibrillary ECM networks. The interactions of glioma cells with the ECM and the degradation of matrix barriers are crucial for both single-cell and collective invasion into neighboring brain tissue. ECM-regulated mechanisms also contribute to immune exclusion, resulting in a major challenge to immunotherapy delivery and efficacy. Glioma cells chemically and physically control the function of their environment, co-opting complex signaling networks for their own benefit, resulting in radio- and chemo-resistance, tumor recurrence, and cancer progression. Targeting these interactions is an attractive strategy for overcoming therapy resistance, and we will discuss recent advances in preclinical studies, current clinical trials, and potential future clinical applications. In this review, we also provide a comprehensive discussion of the complexities of the interconnected cellular and non-cellular components of the microenvironmental landscape of brain tumors to guide the development of safe and effective therapeutic strategies against brain cancer.
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Affiliation(s)
- Syed M. Faisal
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Andrea Comba
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Maria L. Varela
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Anna E. Argento
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI, United States
| | - Emily Brumley
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Clifford Abel
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Maria G. Castro
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
| | - Pedro R. Lowenstein
- Dept. of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States
- Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States
- Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI, United States
- *Correspondence: Pedro R. Lowenstein,
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Zhang Z, Liu C, Yang Z, Yin H. CAR-T-Cell Therapy for Solid Tumors Positive for Fibronectin Extra Domain B. Cells 2022; 11:cells11182863. [PMID: 36139437 PMCID: PMC9496916 DOI: 10.3390/cells11182863] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 09/10/2022] [Accepted: 09/12/2022] [Indexed: 12/24/2022] Open
Abstract
(1) Background: The lack of specific targets has slowed the progress of CAR-T in treating solid tumors. Recent studies have revealed that EDB-FN (fibronectin extra domain B) may be an effective target for CAR-T treatment of solid tumors; EDB-FN is expressed in tumor and embryonic tissues, and antibody–cytokine fusion proteins targeting EDB-FN have been developed. However, the therapeutic effects of BBz CAR-engineered T-cells targeting EDB-FN in solid tumors have not been evaluated. (2) Results: In this study, we constructed a BBz CAR construct targeting EDB-FN, and the CAR molecule was expressed on the surface of T-cells by lentiviral transduction. In vitro, CAR-T-cells can be activated to express perforin and granzyme and lyse EDB-positive cells (U-87 MG cells, A549 cells, and HUVECs) and have no toxicity to EDB-negative cells (MCF-7). Compared to T-cells, CAR-T-cells can release cytokines after coculture with EDB-positive cell lines. In vivo, CAR-T-cells inhibited the progression of U-87 MG subcutaneous tumors and significantly reduced the blood vessel density in tumor tissue compared to T-cells, without obvious toxicity to mouse tissues and organs. Furthermore, CAR-T-cells overexpressing BiTE targeting EDB-FN can significantly improve their antitumor activity in vitro. (3) Conclusions: These results demonstrate that CAR-T-cells have specific antitumor and angiogenic activities in vivo and in vitro, suggesting that EDB-FN may be a potential solid tumor target for CAR-T therapy.
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22
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Nadal L, Peissert F, Elsayed A, Weiss T, Look T, Weller M, Piro G, Carbone C, Tortora G, Matasci M, Favalli N, Corbellari R, Di Nitto C, Prodi E, Libbra C, Galeazzi S, Carotenuto C, Halin C, Puca E, Neri D, De Luca R. Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody. J Immunother Cancer 2022; 10:jitc-2022-005282. [PMID: 36104101 PMCID: PMC9476130 DOI: 10.1136/jitc-2022-005282] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/30/2022] [Indexed: 11/04/2022] Open
Abstract
BACKGROUND In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.
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Affiliation(s)
- Lisa Nadal
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | - Frederik Peissert
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland.,Department of Biology and Biotechnology, IUSS, Pavia, Italy
| | - Abdullah Elsayed
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland.,Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
| | - Tobias Weiss
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | - Thomas Look
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | - Michael Weller
- Department of Neurology and Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland
| | - Geny Piro
- Medical Oncology, Department of Medical and Surgical Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy
| | - Carmine Carbone
- Medical Oncology, Department of Medical and Surgical Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy
| | - Giampaolo Tortora
- Medical Oncology, Department of Medical and Surgical Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy.,Medical Oncology, Department of Translational Medicine, Catholic University of the Sacred Heart, Rome, Italy
| | - Mattia Matasci
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | - Nicholas Favalli
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | | | - Cesare Di Nitto
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | - Eleonora Prodi
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | | | | | | | - Cornelia Halin
- Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
| | - Emanuele Puca
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
| | | | - Roberto De Luca
- Antibody Therapeutics, Philochem AG, Otelfingen, Zurich, Switzerland
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23
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Petrini I, Sollini M, Bartoli F, Barachini S, Montali M, Pardini E, Burzi IS, Erba PA. ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas: A Target for a Theragnostic Approach. Cancers (Basel) 2022; 14:cancers14112592. [PMID: 35681572 PMCID: PMC9179240 DOI: 10.3390/cancers14112592] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Revised: 05/03/2022] [Accepted: 05/16/2022] [Indexed: 11/16/2022] Open
Abstract
Simple Summary The extra-domain B fibronectin (ED-B FN) is highly expressed in thymic epithelial tumors (TETs), as demonstrated by in vivo targeting using 131I-labeled L19 small immunoprotein (131I-L19-SIP) and immunohistochemistry with a predominant expression by stromal cells of a thymoma microenvironment rather than epithelial cells. Such high expression derived from the induction of stromal cells shifts FN production to the ED-B subtype. Our results suggest that Radretumab radioimmunotherapy (R-RIT) inefficacy is not related to low TET ED-B expression but to multifactorial aspects including patients’ inherent characteristics, the pattern expression of the target, the biological characteristics of the tumor, and the format of the target agent, which contribute to the resistance of tumor cells to treatment. Abstract Aim: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). Material and methods: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients’ samples and in the Ty82 cell line. Results: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3–5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. Conclusions: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients’ susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP).
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Affiliation(s)
- Iacopo Petrini
- General Pathology, Department of Translational Research & New Technologies in Surgery and Medicine, University of Pisa and Azienda Ospedaliero Universitaria Pisana, 56100 Pisa, Italy;
| | - Martina Sollini
- Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, 20090 Milan, Italy;
- Diagnostic Imaging Department, IRCCS Humanitas Research Hospital, Rozzano, 20089 Milan, Italy
| | - Francesco Bartoli
- Regional Center of Nuclear Medicine, Department of Translational Research and New Technology in Medicine, University of Pisa and Azienda Ospedaliero Universitaria Pisana, 56126 Pisa, Italy;
| | - Serena Barachini
- Laboratory of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy; (S.B.); (M.M.); (E.P.); (I.S.B.)
| | - Marina Montali
- Laboratory of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy; (S.B.); (M.M.); (E.P.); (I.S.B.)
| | - Eleonora Pardini
- Laboratory of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy; (S.B.); (M.M.); (E.P.); (I.S.B.)
| | - Irene Sofia Burzi
- Laboratory of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy; (S.B.); (M.M.); (E.P.); (I.S.B.)
| | - Paola Anna Erba
- Regional Center of Nuclear Medicine, Department of Translational Research and New Technology in Medicine, University of Pisa and Azienda Ospedaliero Universitaria Pisana, 56126 Pisa, Italy;
- Department of Nuclear Medicine and Molecular Imaging, Medical Imaging Centre, University Medical Center Groningen, 9700 RB Groningen, The Netherlands
- Correspondence: ; Tel.: +39-050-992115
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24
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Oliveira MC, Correia JDG. Clinical application of radioiodinated antibodies: where are we? Clin Transl Imaging 2022. [DOI: 10.1007/s40336-021-00477-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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25
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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: 144] [Impact Index Per Article: 36.0] [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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26
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Uricoli B, Birnbaum LA, Do P, Kelvin JM, Jain J, Costanza E, Chyong A, Porter CC, Rafiq S, Dreaden EC. Engineered Cytokines for Cancer and Autoimmune Disease Immunotherapy. Adv Healthc Mater 2021; 10:e2002214. [PMID: 33690997 PMCID: PMC8651077 DOI: 10.1002/adhm.202002214] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 02/15/2021] [Indexed: 12/17/2022]
Abstract
Cytokine signaling is critical to a range of biological processes including cell development, tissue repair, aging, and immunity. In addition to acting as key signal mediators of the immune system, cytokines can also serve as potent immunotherapies with more than 20 recombinant products currently Food and Drug Administration (FDA)-approved to treat conditions including hepatitis, multiple sclerosis, arthritis, and various cancers. Yet despite their biological importance and clinical utility, cytokine immunotherapies suffer from intrinsic challenges that limit their therapeutic potential including poor circulation, systemic toxicity, and low tissue- or cell-specificity. In the past decade in particular, methods have been devised to engineer cytokines in order to overcome such challenges and here, the myriad strategies are reviewed that may be employed in order to improve the therapeutic potential of cytokine and chemokine immunotherapies with applications in cancer and autoimmune disease therapy, as well as tissue engineering and regenerative medicine. For clarity, these strategies are collected and presented as they vary across size scales, ranging from single amino acid substitutions, to larger protein-polymer conjugates, nano/micrometer-scale particles, and macroscale implants. Together, this work aims to provide readers with a timely view of the field of cytokine engineering with an emphasis on early-stage therapeutic approaches.
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Affiliation(s)
- Biaggio Uricoli
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - Lacey A. Birnbaum
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - Priscilla Do
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - James M. Kelvin
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - Juhi Jain
- Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA
- Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta and Emory School of Medicine, Atlanta, GA 30322, USA
| | - Emma Costanza
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - Andrew Chyong
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
| | - Christopher C. Porter
- Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA
- Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta and Emory School of Medicine, Atlanta, GA 30322, USA
- Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
| | - Sarwish Rafiq
- Department of Hematology and Medical Oncology at Emory University School of Medicine
- Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
| | - Erik C. Dreaden
- Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30322, USA
- Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta and Emory School of Medicine, Atlanta, GA 30322, USA
- Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
- Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA
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27
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Roth KDR, Wenzel EV, Ruschig M, Steinke S, Langreder N, Heine PA, Schneider KT, Ballmann R, Fühner V, Kuhn P, Schirrmann T, Frenzel A, Dübel S, Schubert M, Moreira GMSG, Bertoglio F, Russo G, Hust M. Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy. Front Cell Infect Microbiol 2021; 11:697876. [PMID: 34307196 PMCID: PMC8294040 DOI: 10.3389/fcimb.2021.697876] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 06/21/2021] [Indexed: 12/30/2022] Open
Abstract
Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.
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Affiliation(s)
- Kristian Daniel Ralph Roth
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Esther Veronika Wenzel
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany
| | - Maximilian Ruschig
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Stephan Steinke
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Nora Langreder
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Philip Alexander Heine
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Kai-Thomas Schneider
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Rico Ballmann
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Viola Fühner
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | | | | | | | - Stefan Dübel
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany.,YUMAB GmbH, Braunschweig, Germany
| | - Maren Schubert
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | | | - Federico Bertoglio
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Giulio Russo
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany
| | - Michael Hust
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,YUMAB GmbH, Braunschweig, Germany
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28
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Corbellari R, Stringhini M, Mock J, Ongaro T, Villa A, Neri D, De Luca R. A Novel Antibody-IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis. Mol Cancer Ther 2021; 20:859-871. [PMID: 33632875 DOI: 10.1158/1535-7163.mct-20-0853] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 01/14/2021] [Accepted: 02/09/2021] [Indexed: 12/23/2022]
Abstract
IL15 is an immunostimulatory cytokine that holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor α, capable of binding to IL15) has been fused to IL15-based anticancer products to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematologic tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted TNF. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.
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Affiliation(s)
- Riccardo Corbellari
- CiBIO (Department of Cellular, Computational and Integrative Biology), University of Trento, Povo, Trento, Italy.,Philochem AG, Otelfingen, Switzerland
| | - Marco Stringhini
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zurich, Switzerland
| | - Jaqueline Mock
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zurich, Switzerland
| | | | | | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zurich, Switzerland
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29
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Campbell SM, DeBartolo J, Apgar JR, Mosyak L, McManus V, Beyer S, Bennett EM, Lambert M, Cunningham O. Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate polyreactivity of an anti-IL-21R antibody. MAbs 2021; 13:1883239. [PMID: 33557673 PMCID: PMC7889167 DOI: 10.1080/19420862.2021.1883239] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Despite substantial technological advances in antibody library and display platform development, the number of approved biotherapeutics from displayed libraries remains limited. In vivo, 20–50% of peripheral B cells undergo a process of receptor editing, which modifies the variable and junctional regions of light chains to delete auto-reactive clones. However, in vitro antibody evolution relies primarily on interaction with antigen, with no in-built checkpoints to ensure that the selected antibodies have not acquired additional specificities or biophysical liabilities during the optimization process. We had previously observed an enrichment of positive charge in the complementarity-determining regions of an anti-IL-21 R antibody during affinity optimization, which correlated with more potent IL-21 neutralization, but poor in vivo pharmacokinetics (PK). There is an emerging body of data that has correlated antibody nonspecificity with poor PK in vivo, and established a series of screening assays that are predictive of this behavior. In this study we revisit the challenge of developing an anti-IL-21 R antibody that can effectively compete with IL-21 for its highly negatively charged paratope while maintaining favorable biophysical properties. In vitro deselection methods that included an excess of negatively charged membrane preparations, or deoxyribonucleic acid, during phage selection of optimization libraries were unsuccessful in avoiding enrichment of highly charged, nonspecific antibody variants. However, a combination of structure-guided rational library design, next-generation sequencing of library outputs and application of linear regression models resulted in the identification of an antibody that maintained high affinity for IL-21 R and exhibited a desirable stability and biophysical profile.
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Affiliation(s)
| | | | | | | | | | - Sonia Beyer
- Biomedicine Design, Pfizer , Dublin, Ireland
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30
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Ongaro T, Guarino SR, Scietti L, Palamini M, Wulhfard S, Neri D, Villa A, Forneris F. Inference of molecular structure for characterization and improvement of clinical grade immunocytokines. J Struct Biol 2021; 213:107696. [PMID: 33493635 DOI: 10.1016/j.jsb.2021.107696] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2020] [Revised: 12/14/2020] [Accepted: 01/11/2021] [Indexed: 10/22/2022]
Abstract
The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.
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Affiliation(s)
- Tiziano Ongaro
- The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy; Philochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland
| | - Salvatore R Guarino
- The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy
| | - Luigi Scietti
- The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy
| | - Martina Palamini
- The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy
| | - Sarah Wulhfard
- Philochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093 Zürich Switzerland
| | | | - Federico Forneris
- The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy.
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31
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Garcia-Calvo E, García-García A, Madrid R, Martin R, García T. From Polyclonal Sera to Recombinant Antibodies: A Review of Immunological Detection of Gluten in Foodstuff. Foods 2020; 10:foods10010066. [PMID: 33396828 PMCID: PMC7824297 DOI: 10.3390/foods10010066] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2020] [Revised: 12/23/2020] [Accepted: 12/24/2020] [Indexed: 12/31/2022] Open
Abstract
Gluten is the ethanol-soluble protein fraction of cereal endosperms like wheat, rye, and barley. It is widely used in the food industry because of the physical-chemical properties it gives to dough. Nevertheless, there are some gluten-related diseases that are presenting increasing prevalences, e.g., celiac disease, for which a strict gluten-free diet is the best treatment. Due to this situation, gluten labeling legislation has been developed in several countries around the world. This article reviews the gluten immune detection systems that have been applied to comply with such regulations. These systems have followed the development of antibody biotechnology, which comprise three major methodologies: polyclonal antibodies, monoclonal antibodies (mAbs) derived from hybridoma cells (some examples are 401.21, R5, G12, and α-20 antibodies), and the most recent methodology of recombinant antibodies. Initially, the main objective was the consecution of new high-affinity antibodies, resulting in low detection and quantification limits that are mainly achieved with the R5 mAb (the gold standard for gluten detection). Increasing knowledge about the causes of gluten-related diseases has increased the complexity of research in this field, with current efforts not only focusing on the development of more specific and sensitive systems for gluten but also the detection of protein motifs related to pathogenicity. New tools based on recombinant antibodies will provide adequate safety and traceability methodologies to meet the increasing market demand for gluten-free products.
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32
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Wagner J, Wickman E, Shaw TI, Anido AA, Langfitt D, Zhang J, Porter SN, Pruett-Miller SM, Tillman H, Krenciute G, Gottschalk S. Antitumor Effects of CAR T Cells Redirected to the EDB Splice Variant of Fibronectin. Cancer Immunol Res 2020; 9:279-290. [DOI: 10.1158/2326-6066.cir-20-0280] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Revised: 10/19/2020] [Accepted: 12/09/2020] [Indexed: 11/16/2022]
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33
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Nadal L, Corbellari R, Villa A, Weiss T, Weller M, Neri D, De Luca R. Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors in vivo. MAbs 2020; 12:1836713. [PMID: 33136526 PMCID: PMC7646483 DOI: 10.1080/19420862.2020.1836713] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients.
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Affiliation(s)
- Lisa Nadal
- Biology department, Philochem AG , Otelfingen, Switzerland.,CiBIO (Department of Cellular, Computational and Integrative Biology, University of Trento, Italy , Trento, Italy
| | - Riccardo Corbellari
- Biology department, Philochem AG , Otelfingen, Switzerland.,CiBIO (Department of Cellular, Computational and Integrative Biology, University of Trento, Italy , Trento, Italy
| | | | - Tobias Weiss
- Department of Neurology and Brain Tumor Center, University Hospital Zurich and University of Zurich , Zurich, Switzerland
| | - Michael Weller
- Department of Neurology and Brain Tumor Center, University Hospital Zurich and University of Zurich , Zurich, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich) , Zurich, Switzerland
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34
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Mortensen MR, Mock J, Bertolini M, Stringhini M, Catalano M, Neri D. Targeting an engineered cytokine with interleukin-2 and interleukin-15 activity to the neovasculature of solid tumors. Oncotarget 2020; 11:3972-3983. [PMID: 33216834 PMCID: PMC7646832 DOI: 10.18632/oncotarget.27772] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Accepted: 09/24/2020] [Indexed: 01/07/2023] Open
Abstract
There is a growing interest in the antibody-based delivery of cytokines to the tumor environment as a means to boost the anti-cancer activity of tumor-resident T cells and NK cells. Here, we describe the expression and characterization of fusion proteins, featuring the L19 antibody (specific to the alternatively-spliced EDB domain of fibronectin) and an engineered cytokine with interleukin-2 and interleukin-15 properties. The cytokine moiety was fused either at the N-terminal or at the C-terminal extremity and both fusion proteins showed a selective tumor accumulation in a quantitative biodistribution experiment. The N-terminal fusion inhibited tumor growth in immunocompetent mice bearing F9 carcinomas or WEHI-164 sarcomas when used as single agent. The anticancer activity was compared to the one of the same cytokine payload used as recombinant protein or fused to an anti-hen egg lysozyme antibody, serving as negative control of irrelevant specificity in the mouse. These results indicate that the antibody-based delivery of engineered cytokines to the tumor neovasculature may mediate a potent anticancer activity.
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Affiliation(s)
- Michael R Mortensen
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
| | - Jacqueline Mock
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
| | - Marco Bertolini
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
| | - Marco Stringhini
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
| | - Marco Catalano
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland
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35
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A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo. Oncotarget 2020; 11:3698-3711. [PMID: 33110477 PMCID: PMC7566808 DOI: 10.18632/oncotarget.27726] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2020] [Accepted: 08/17/2020] [Indexed: 01/13/2023] Open
Abstract
The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.
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36
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Weishaupt C, Goerge T, Loser K. Activated melanoma vessels: A sticky point for successful immunotherapy. Exp Dermatol 2020; 29:1046-1054. [PMID: 32998178 DOI: 10.1111/exd.14203] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2020] [Revised: 09/07/2020] [Accepted: 09/21/2020] [Indexed: 11/30/2022]
Abstract
Metastatic melanoma is a devastating disease with a marginal-albeit increasing-hope for cure. Melanoma has a high mutation rate which correlates to the expression of numerous neo-antigens and thus is associated with the potential to induce and strengthen effective antitumoral immunity. However, the incomplete and potentially insufficient response to established immunotherapies (response rates usually do not markedly exceed 60%) already points to the need of further studies to improve treatment strategies. Multiple tumor escape mechanisms that allow melanoma to evade from antitumoral immune responses have been characterized and must be overcome to achieve a better clinical efficacy of immunotherapies. Recently, promising progress has been made in targeting tumor vasculature to control and increase the infiltration of tumors with effector lymphocytes. It has been hypothesized that amplified lymphocytic infiltrates in melanoma metastases result in a switch of the tumor microenvironment from a non-inflammatory to an inflammatory state. In this view point essay, we discuss the requirements for successful homing of lymphocytes to melanoma tissue and we present a mouse melanoma xenograft model that allows the investigation of human tumor vessels in vivo. Furthermore, current clinical studies dealing with the activation of melanoma vasculature for enhanced effectiveness of immunotherapy protocols are presented and open questions for routine clinical application are addressed.
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Affiliation(s)
- Carsten Weishaupt
- Department of Dermatology, University Hospital of Muenster, Muenster, Germany
| | - Tobias Goerge
- Department of Dermatology, University Hospital of Muenster, Muenster, Germany
| | - Karin Loser
- Department of Dermatology, University Hospital of Muenster, Muenster, Germany.,Institute of Immunology, University of Oldenburg, Oldenburg, Germany
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37
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Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, Hashem AM. Phage Display Derived Monoclonal Antibodies: From Bench to Bedside. Front Immunol 2020; 11:1986. [PMID: 32983137 PMCID: PMC7485114 DOI: 10.3389/fimmu.2020.01986] [Citation(s) in RCA: 162] [Impact Index Per Article: 32.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Accepted: 07/23/2020] [Indexed: 12/12/2022] Open
Abstract
Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage–derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.
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Affiliation(s)
- Mohamed A Alfaleh
- Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia.,Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Hashem O Alsaab
- Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taif University, Taif, Saudi Arabia
| | - Ahmad Bakur Mahmoud
- College of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia
| | - Almohanad A Alkayyal
- Department of Medical Laboratory Technology, University of Tabuk, Tabuk, Saudi Arabia
| | - Martina L Jones
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia.,Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, QLD, Australia
| | - Stephen M Mahler
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia.,Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, QLD, Australia
| | - Anwar M Hashem
- Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia.,Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
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38
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Epitope Mapping and Computational Analysis of Anti-HPV16 E6 and E7 Antibodies in Single-Chain Format for Clinical Development as Antitumor Drugs. Cancers (Basel) 2020; 12:cancers12071803. [PMID: 32640530 PMCID: PMC7408665 DOI: 10.3390/cancers12071803] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Revised: 07/02/2020] [Accepted: 07/03/2020] [Indexed: 11/17/2022] Open
Abstract
Human Papillomavirus 16-associated cancer, affecting primarily the uterine cervix but, increasingly, other body districts, including the head–neck area, will long be a public health problem, despite there being a vaccine. Since the virus oncogenic activity is fully ascribed to the viral E6 and E7 oncoproteins, one of the therapeutic approaches for HPV16 cancer is based on specific antibodies in single-chain format targeting the E6/E7 activity. We analyzed the Complementarity Determining Regions, repositories of antigen-binding activity, of four anti-HPV16 E6 and -HPV16 E7 scFvs, to highlight possible conformity to biophysical properties, recognized to be advantageous for therapeutic use. By epitope mapping, using E7 mutants with amino acid deletions or variations, we investigated differences among the anti-16E7 scFvs in terms of antigen-binding capacity. We also performed computational analyses to determine whether length, total net charge, surface hydrophobicity, polarity and charge distribution conformed well to those of the antibodies that had already reached clinical use, through the application of developability guidelines derived from recent literature on clinical-stage antibodies, and the Therapeutic Antibodies Profiler software. Overall, our findings show that the scFvs investigated may represent valid candidates to be developed as therapeutic molecules for clinical use, and highlight characteristics that could be improved by molecular engineering.
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39
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Lieverse RIY, Marcus D, van der Wiel AMA, Van Limbergen EJ, Theys J, Yaromina A, Lambin P, Dubois LJ. Human fibronectin extra domain B as a biomarker for targeted therapy in cancer. Mol Oncol 2020; 14:1555-1568. [PMID: 32386436 PMCID: PMC7332215 DOI: 10.1002/1878-0261.12705] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 04/15/2020] [Accepted: 05/04/2020] [Indexed: 12/12/2022] Open
Abstract
The extracellular matrix protein fibronectin contains a domain that is rarely found in healthy adults and is almost exclusively expressed by newly formed blood vessels in tumours, particularly in solid tumours, different types of lymphoma and some leukaemias. This domain, called the extra domain B (ED‐B), thus has broad therapeutic potential. The antibody L19 has been developed to specifically target ED‐B and has shown therapeutic potential when combined with cytokines, such as IL‐2. In this review article, we discuss the preclinical research and clinical trials that highlight the potential of ED‐B targeting for the imaging and treatment of various types of cancer. ED‐B‐centred studies also highlight how proper patient stratification is of utmost importance for the successful implementation of novel antibody‐based targeted therapies.
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Affiliation(s)
- Relinde I Y Lieverse
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Damiënne Marcus
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Alexander M A van der Wiel
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Evert J Van Limbergen
- Department of Radiation Oncology (MAASTRO), GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands
| | - Jan Theys
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Ala Yaromina
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Philippe Lambin
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
| | - Ludwig J Dubois
- The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, The Netherlands
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40
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Neri D. Antibody-Cytokine Fusions: Versatile Products for the Modulation of Anticancer Immunity. Cancer Immunol Res 2020; 7:348-354. [PMID: 30824549 DOI: 10.1158/2326-6066.cir-18-0622] [Citation(s) in RCA: 96] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The remarkable clinical success of immune-checkpoint inhibitors for the treatment of a growing number of cancer types has sparked interest in the discovery of novel forms of immunotherapy, which may be used alone or in combination. In this context, cytokine-based therapeutics are well poised to play a role in modern cancer therapy. This article focuses on antibody-cytokine fusion proteins (also called "immunocytokines") as one class of biopharmaceuticals that can substantially improve the therapeutic index and, thus, the applicability of cytokine products. In many preclinical settings, antibodies can be used to preferentially deliver many (but not all) types of cytokines to primary and metastatic tumor lesions. The antibody-based delivery of certain proinflammatory payloads (such as IL2, IL12, and TNF) to the tumor microenvironment can lead to a dramatic potentiation of their anticancer activity. However, although some fusion proteins have advanced to late-stage clinical trials, much work remains to be done in order to fully characterize the mechanism of action and the pharmaceutical potential of immunocytokines in the clinical setting. Various factors contribute to in vivo performance, including the target antigen, the antibody properties, the nature of the payload, the format of the fusion protein, the dose, and schedule, as well as their use in combination with other therapeutic modalities. Protein engineering opportunities and insights in cancer immunology are contributing to the development of next-generation immunocytokine products and of novel therapeutic concepts, with the goal to increase antitumor activity and reduce systemic toxicity (a common problem for cytokine-based biopharmaceuticals).
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Affiliation(s)
- Dario Neri
- Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, Switzerland.
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41
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Mock J, Pellegrino C, Neri D. A universal reporter cell line for bioactivity evaluation of engineered cytokine products. Sci Rep 2020; 10:3234. [PMID: 32094407 PMCID: PMC7040017 DOI: 10.1038/s41598-020-60182-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Accepted: 12/07/2019] [Indexed: 12/24/2022] Open
Abstract
Engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. In most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. The novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-κB. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed “universal” bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-κB were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. The newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.
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Affiliation(s)
- Jacqueline Mock
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland
| | - Christian Pellegrino
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland.
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42
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Lui BG, Salomon N, Wüstehube-Lausch J, Daneschdar M, Schmoldt HU, Türeci Ö, Sahin U. Targeting the tumor vasculature with engineered cystine-knot miniproteins. Nat Commun 2020; 11:295. [PMID: 31941901 PMCID: PMC6962393 DOI: 10.1038/s41467-019-13948-y] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Accepted: 11/28/2019] [Indexed: 01/08/2023] Open
Abstract
The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications. Cystine-knot miniprotein are small, highly stable, disulfide-rich peptides with increasing potential as drugs and tumor imaging agents. Here the authors develop cystine-knot miniproteins targeting the vascular tumor marker EDB, and use them as probes for in vivo tumor vasculature imaging.
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43
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Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, Hashem AM. Phage Display Derived Monoclonal Antibodies: From Bench to Bedside. Front Immunol 2020. [PMID: 32983137 DOI: 10.3389/fimmu.2020.01986/bibtex] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/05/2023] Open
Abstract
Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage-derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.
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Affiliation(s)
- Mohamed A Alfaleh
- Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
- Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Hashem O Alsaab
- Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taif University, Taif, Saudi Arabia
| | - Ahmad Bakur Mahmoud
- College of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia
| | - Almohanad A Alkayyal
- Department of Medical Laboratory Technology, University of Tabuk, Tabuk, Saudi Arabia
| | - Martina L Jones
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia
- Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, QLD, Australia
| | - Stephen M Mahler
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia
- Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, QLD, Australia
| | - Anwar M Hashem
- Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
- Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
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Abstract
Single photon emission computed tomography (SPECT) is the state-of-the-art imaging modality in nuclear medicine despite the fact that only a few new SPECT tracers have become available in the past 20 years. Critical for the future success of SPECT is the design of new and specific tracers for the detection, localization, and staging of a disease and for monitoring therapy. The utility of SPECT imaging to address oncologic questions is dependent on radiotracers that ideally exhibit excellent tissue penetration, high affinity to the tumor-associated target structure, specific uptake and retention in the malignant lesions, and rapid clearance from non-targeted tissues and organs. In general, a target-specific SPECT radiopharmaceutical can be divided into two main parts: a targeting biomolecule (e.g., peptide, antibody fragment) and a γ-radiation-emitting radionuclide (e.g., 99mTc, 123I). If radiometals are used as the radiation source, a bifunctional chelator is needed to link the radioisotope to the targeting entity. In a rational SPECT tracer design, these single components have to be critically evaluated in order to achieve a balance among the demands for adequate target binding, and a rapid clearance of the radiotracer. The focus of this chapter is to depict recent developments of tumor-targeted SPECT radiotracers for imaging of cancer diseases. Possibilities for optimization of tracer design and potential causes for design failure are discussed and highlighted with selected examples.
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Flego M, Frau A, Accardi L, Mallano A, Ascione A, Gellini M, Fanunza E, Vella S, Di Bonito P, Tramontano E. Intracellular human antibody fragments recognizing the VP35 protein of Zaire Ebola filovirus inhibit the protein activity. BMC Biotechnol 2019; 19:64. [PMID: 31488108 PMCID: PMC6727353 DOI: 10.1186/s12896-019-0554-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2019] [Accepted: 08/09/2019] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. RESULTS Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35. CONCLUSION Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.
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Affiliation(s)
- Michela Flego
- Istituto Superiore di Sanità (ISS), National Center for Global Health, Viale Regina Elena 299, 00161, Rome, Italy.
| | - Aldo Frau
- Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SS554 09042 Monserrato, Cagliari, Italy
| | - Luisa Accardi
- Department of Infectious Diseases, Viral Hepatitis, Oncoviruses and Retroviruses (EVOR) unit, Istituto Superiore di Sanità (ISS), Viale Regina Elena 299, 00161, Rome, Italy
| | - Alessandra Mallano
- Istituto Superiore di Sanità (ISS), National Center for Global Health, Viale Regina Elena 299, 00161, Rome, Italy
| | - Alessandro Ascione
- Istituto Superiore di Sanità (ISS), National Center for Global Health, Viale Regina Elena 299, 00161, Rome, Italy
| | - Mara Gellini
- Istituto Superiore di Sanità (ISS), National Center for Global Health, Viale Regina Elena 299, 00161, Rome, Italy
| | - Elisa Fanunza
- Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SS554 09042 Monserrato, Cagliari, Italy
| | - Stefano Vella
- Istituto Superiore di Sanità (ISS), National Center for Global Health, Viale Regina Elena 299, 00161, Rome, Italy
| | - Paola Di Bonito
- Department of Infectious Diseases, Viral Hepatitis, Oncoviruses and Retroviruses (EVOR) unit, Istituto Superiore di Sanità (ISS), Viale Regina Elena 299, 00161, Rome, Italy.
| | - Enzo Tramontano
- Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SS554 09042 Monserrato, Cagliari, Italy.
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Puca E, Probst P, Stringhini M, Murer P, Pellegrini G, Cazzamalli S, Hutmacher C, Gouyou B, Wulhfard S, Matasci M, Villa A, Neri D. The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8 + T cell activity and synergizes with immune checkpoint inhibitors. Int J Cancer 2019; 146:2518-2530. [PMID: 31374124 DOI: 10.1002/ijc.32603] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Revised: 07/03/2019] [Accepted: 07/17/2019] [Indexed: 02/06/2023]
Abstract
We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.
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Affiliation(s)
- Emanuele Puca
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
| | - Philipp Probst
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
| | - Marco Stringhini
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
| | - Patrizia Murer
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
| | - Giovanni Pellegrini
- Laboratory for Animal Model Pathology, Universität Zürich, Zürich, Switzerland
| | | | - Cornelia Hutmacher
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
| | | | | | | | | | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland
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Almagro JC, Pedraza-Escalona M, Arrieta HI, Pérez-Tapia SM. Phage Display Libraries for Antibody Therapeutic Discovery and Development. Antibodies (Basel) 2019; 8:antib8030044. [PMID: 31544850 PMCID: PMC6784186 DOI: 10.3390/antib8030044] [Citation(s) in RCA: 97] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2019] [Revised: 08/14/2019] [Accepted: 08/15/2019] [Indexed: 01/24/2023] Open
Abstract
Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.
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Affiliation(s)
- Juan C Almagro
- GlobalBio, Inc., 320, Cambridge, MA 02138, USA.
- UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico.
| | - Martha Pedraza-Escalona
- CONACyT-UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico
| | - Hugo Iván Arrieta
- CONACyT-UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico
| | - Sonia Mayra Pérez-Tapia
- CONACyT-UDIBI, ENCB, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Casco de Santo Tomas, Delegación Miguel Hidalgo, Ciudad de Mexico 11340, Mexico
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Hutmacher C, Volta L, Rinaldi F, Murer P, Myburgh R, Manz MG, Neri D. Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia. Leuk Res 2019; 84:106178. [PMID: 31326578 DOI: 10.1016/j.leukres.2019.106178] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2019] [Revised: 06/24/2019] [Accepted: 06/27/2019] [Indexed: 10/26/2022]
Abstract
Monoclonal antibodies are being considered as biopharmaceuticals for the in vivo targeting of acute myeloid leukemia. Here we describe the generation and characterization of a fully-human monoclonal antibody specific to CD123, a surface marker which is overexpressed in a variety of hematological disorders, including acute myeloid leukemia. The cloning and expression of the extracellular portion of CD123 as recombinant Fc fusion allowed the selection and affinity maturation of a human antibody, called H9, which specifically recognized the cognate antigen in biochemical assays and on leukemic cells. The H9 antibody and a previously-described anti-CD123 antibody (CSL362) were reformatted into full immunoglobulin human IgG1 formats, including a variant bearing S293D and I332E mutations to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). The two antibodies recognized different epitopes on the surface of the N-terminal domain of CD123, as revealed by crystallography and SPOT analysis. Both H9 and CSL362 in full immunoglobulin format were able to selectively kill leukemic cells in in vitro ADCC assays, performed both with cell lines and with patient-derived AML blasts. Further, the two antibodies, when reformatted as bispecific BiTE™ reagents by fusion with the anti-CD3 scFv(OKT3) antibody fragment, induced selective killing of AML blasts by patient-derived, autologous T-cells in an in vitro setting, but BiTE(CSL362/OKT3) exhibited a 10-fold higher potency compared to BiTE(H9/OKT3). The availability of two classes of CD123-specific biopharmaceuticals, capable of redirecting the cytolytic activity of NK cells and T cells against AML blasts, may enable novel interventional strategies and combination opportunities for the treatment of AML.
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Affiliation(s)
- Cornelia Hutmacher
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Vladimir-Prelog Weg 1-5/10, CH-8093 Zurich, Switzerland
| | - Laura Volta
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Vladimir-Prelog Weg 1-5/10, CH-8093 Zurich, Switzerland
| | - Francesco Rinaldi
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Vladimir-Prelog Weg 1-5/10, CH-8093 Zurich, Switzerland
| | - Patrizia Murer
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Vladimir-Prelog Weg 1-5/10, CH-8093 Zurich, Switzerland
| | - Renier Myburgh
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland
| | - Markus G Manz
- Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Vladimir-Prelog Weg 1-5/10, CH-8093 Zurich, Switzerland.
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Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K. Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol 2019; 135:907-918. [PMID: 31170490 DOI: 10.1016/j.ijbiomac.2019.06.006] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Revised: 06/02/2019] [Accepted: 06/02/2019] [Indexed: 12/29/2022]
Abstract
Monoclonal antibodies (mAbs) and their derivatives have achieved remarkable success as medicine, targeting both diagnostic and therapeutic applications associated with communicable and non-communicable diseases. In the last 3 to 4 decades, tremendous success has been manifested in the field of cancer therapy, autoimmune diseases, cardiovascular and infectious diseases. MAbs are the fastest growing class of biopharmaceuticals, with more than 25 derivatives are in clinical use and 7 of these have been isolated through phage display technology. Phage display technology has gained impetus in the field of medical and health sciences, as a large repertoire of diverse recombinant antibodies, targeting various antigens have been generated in a short span of time. A prominent number of phage display derived antibodies are already approved for therapy and significant numbers are currently in clinical trials. In this review we have discussed the various strategies employed for generation of monoclonal antibodies; their advantages, limitations and potential therapeutic applications. We also discuss the potential of phage display antibody libraries in isolation of monoclonal antibodies.
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Affiliation(s)
- Rajesh Kumar
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India; Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India.
| | - Hilal Ahmed Parray
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
| | - Tripti Shrivastava
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Subrata Sinha
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
| | - Kalpana Luthra
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
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Lim CC, Choong YS, Lim TS. Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation. Int J Mol Sci 2019; 20:E1861. [PMID: 30991723 PMCID: PMC6515083 DOI: 10.3390/ijms20081861] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 04/10/2019] [Accepted: 04/12/2019] [Indexed: 12/25/2022] Open
Abstract
Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some 'fine tuning' may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.
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Affiliation(s)
- Chia Chiu Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia.
| | - Yee Siew Choong
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia.
| | - Theam Soon Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia.
- Analytical Biochemistry Research Centre, Universiti Sains Malaysia, Penang 11800, Malaysia.
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