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Liu J, Wu L, Xie A, Liu W, He Z, Wan Y, Mao W. Unveiling the new chapter in nanobody engineering: advances in traditional construction and AI-driven optimization. J Nanobiotechnology 2025; 23:87. [PMID: 39915791 PMCID: PMC11800653 DOI: 10.1186/s12951-025-03169-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2024] [Accepted: 01/27/2025] [Indexed: 02/11/2025] Open
Abstract
Nanobodies (Nbs), miniature antibodies consisting solely of the variable region of heavy chains, exhibit unique properties such as small size, high stability, and strong specificity, making them highly promising for disease diagnosis and treatment. The engineering production of Nbs has evolved into a mature process, involving library construction, screening, and expression purification. Different library types, including immune, naïve, and synthetic/semi-synthetic libraries, offer diverse options for various applications, while display platforms like phage display, cell surface display, and non-surface display provide efficient screening of target Nbs. Recent advancements in artificial intelligence (AI) have opened new avenues in Nb engineering. AI's exceptional performance in protein structure prediction and molecular interaction simulation has introduced novel perspectives and tools for Nb design and optimization. Integrating AI with traditional experimental methods is anticipated to enhance the efficiency and precision of Nb development, expediting the transition from basic research to clinical applications. This review comprehensively examines the latest progress in Nb engineering, emphasizing library construction strategies, display platform technologies, and AI applications. It evaluates the strengths and weaknesses of various libraries and display platforms and explores the potential and challenges of AI in predicting Nb structure, antigen-antibody interactions, and optimizing physicochemical properties.
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Affiliation(s)
- Jiwei Liu
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China
- Wuxi College of Clinical Medicine, Nanjing Medical University, Wuxi, 214023, China
| | - Lei Wu
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China
- Wuxi College of Clinical Medicine, Nanjing Medical University, Wuxi, 214023, China
| | - Anqi Xie
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China
| | - Weici Liu
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China
- Wuxi College of Clinical Medicine, Nanjing Medical University, Wuxi, 214023, China
| | - Zhao He
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China
- Wuxi College of Clinical Medicine, Nanjing Medical University, Wuxi, 214023, China
| | - Yuan Wan
- The Pq Laboratory of BiomeDx/Rx, Department of Biomedical Engineering, Binghamton University, Binghamton, 13850, USA.
- Department of Biomedical Engineering, The Pq Laboratory of BiomeDx/Rx, Binghamton University, Binghamton, NY, 13902, USA.
| | - Wenjun Mao
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, Wuxi, 214023, China.
- Wuxi College of Clinical Medicine, Nanjing Medical University, Wuxi, 214023, China.
- Department of Thoracic Surgery, Wuxi People's Hospital, Wuxi Medical Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Nanjing Medical University, No. 299 Qingyang Rd., Wuxi, 214023, China.
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Kassardjian A, Ivanochko D, Barber B, Jetha A, Julien JP. Humanization of Pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework. Antibodies (Basel) 2024; 13:57. [PMID: 39051333 PMCID: PMC11270187 DOI: 10.3390/antib13030057] [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: 05/21/2024] [Revised: 07/10/2024] [Accepted: 07/11/2024] [Indexed: 07/27/2024] Open
Abstract
Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines.
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Affiliation(s)
- Audrey Kassardjian
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada
- Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
| | - Danton Ivanochko
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Brian Barber
- Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
| | - Arif Jetha
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Jean-Philippe Julien
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada
- Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
- Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
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Wang M, Ying T, Wu Y. Single-domain antibodies as therapeutics for solid tumor treatment. Acta Pharm Sin B 2024; 14:2854-2868. [PMID: 39027249 PMCID: PMC11252471 DOI: 10.1016/j.apsb.2024.03.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 02/23/2024] [Accepted: 03/01/2024] [Indexed: 07/20/2024] Open
Abstract
Single-domain antibodies (sdAbs), initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs, have emerged as a promising alternative to conventional therapeutic antibodies. These sdAbs have many superior physicochemical and pharmacological properties, including small size, good solubility and thermostability, easier accessible epitopes, and strong tissue penetration. However, the inherent challenges associated with the animal origin of sdAbs limit their clinical use. In recent years, various innovative humanization technologies, including complementarity-determining region (CDR) grafting or complete engineering of fully human sdAbs, have been developed to mitigate potential immunogenicity issues and enhance their compatibility. This review provides a comprehensive exploration of sdAbs, emphasizing their distinctive features and the progress in humanization methodologies. In addition, we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment, such as sdAb-drug conjugates, multispecific sdAbs, sdAb-based delivery systems, and sdAb-based cell therapy.
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Affiliation(s)
- Mingkai Wang
- MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Department of Pulmonary and Critical Care Medicine, Department of Liver Surgery and Transplantation, Zhongshan Hospital, Fudan University, Shanghai 200032, China
- Shanghai Engineering Research Center for Synthetic Immunology, Shanghai 200032, China
| | - Tianlei Ying
- MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Department of Pulmonary and Critical Care Medicine, Department of Liver Surgery and Transplantation, Zhongshan Hospital, Fudan University, Shanghai 200032, China
- Shanghai Engineering Research Center for Synthetic Immunology, Shanghai 200032, China
| | - Yanling Wu
- MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Department of Pulmonary and Critical Care Medicine, Department of Liver Surgery and Transplantation, Zhongshan Hospital, Fudan University, Shanghai 200032, China
- Shanghai Engineering Research Center for Synthetic Immunology, Shanghai 200032, China
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Gordon GL, Raybould MIJ, Wong A, Deane CM. Prospects for the computational humanization of antibodies and nanobodies. Front Immunol 2024; 15:1399438. [PMID: 38812514 PMCID: PMC11133524 DOI: 10.3389/fimmu.2024.1399438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2024] [Accepted: 05/02/2024] [Indexed: 05/31/2024] Open
Abstract
To be viable therapeutics, antibodies must be tolerated by the human immune system. Rational approaches to reduce the risk of unwanted immunogenicity involve maximizing the 'humanness' of the candidate drug. However, despite the emergence of new discovery technologies, many of which start from entirely human gene fragments, most antibody therapeutics continue to be derived from non-human sources with concomitant humanization to increase their human compatibility. Early experimental humanization strategies that focus on CDR loop grafting onto human frameworks have been critical to the dominance of this discovery route but do not consider the context of each antibody sequence, impacting their success rate. Other challenges include the simultaneous optimization of other drug-like properties alongside humanness and the humanization of fundamentally non-human modalities such as nanobodies. Significant efforts have been made to develop in silico methodologies able to address these issues, most recently incorporating machine learning techniques. Here, we outline these recent advancements in antibody and nanobody humanization, focusing on computational strategies that make use of the increasing volume of sequence and structural data available and the validation of these tools. We highlight that structural distinctions between antibodies and nanobodies make the application of antibody-focused in silico tools to nanobody humanization non-trivial. Furthermore, we discuss the effects of humanizing mutations on other essential drug-like properties such as binding affinity and developability, and methods that aim to tackle this multi-parameter optimization problem.
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Affiliation(s)
| | | | | | - Charlotte M. Deane
- Oxford Protein Informatics Group, Department of Statistics, University of Oxford, Oxford, United Kingdom
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Wang S, Wang H, Drabek A, Smith WS, Liang F, Huang ZR. Unleashing the Potential: Designing Antibody-Targeted Lipid Nanoparticles for Industrial Applications with CMC Considerations and Clinical Outlook. Mol Pharm 2024; 21:4-17. [PMID: 38117251 DOI: 10.1021/acs.molpharmaceut.3c00735] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2023]
Abstract
Antibody-targeted lipid nanoparticles (Ab-LNPs) are rapidly gaining traction as multifaceted platforms in precision medicine, adept at delivering a diverse array of therapeutic agents, including nucleic acids and small molecules. This review provides an incisive overview of the latest developments in the field of Ab-LNP technology, with a special emphasis on pivotal design aspects such as antibody engineering, bioconjugation strategies, and advanced formulation techniques. Furthermore, it addresses critical chemistry, manufacturing, and controls (CMC) considerations and thoroughly examines the in vivo dynamics of Ab-LNPs, underscoring their promising potential for clinical application. By seamlessly blending scientific advancements with practical industrial perspectives, this review casts a spotlight on the burgeoning role of Ab-LNPs as an innovative and potent tool in the realm of targeted drug delivery.
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Affiliation(s)
- Sheryl Wang
- Sanofi, Genomic Medicine Unit, 225 Second Avenue, Waltham, Massachusetts 02451, United States
| | - Hong Wang
- Sanofi, Genomic Medicine Unit, 225 Second Avenue, Waltham, Massachusetts 02451, United States
| | - Andrew Drabek
- Sanofi, Genomic Medicine Unit, 225 Second Avenue, Waltham, Massachusetts 02451, United States
| | - Wenwen Sha Smith
- FUSION BioVenture, 15 Presidential Way, Woburn, Massachusetts 01801, United States
| | - Feng Liang
- Sanofi, Genomic Medicine Unit, 225 Second Avenue, Waltham, Massachusetts 02451, United States
| | - Zhaohua Richard Huang
- Sanofi, Genomic Medicine Unit, 225 Second Avenue, Waltham, Massachusetts 02451, United States
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Mo G, Lu X, Wu S, Zhu W. Strategies and rules for tuning TCR-derived therapy. Expert Rev Mol Med 2023; 26:e4. [PMID: 38095091 PMCID: PMC11062142 DOI: 10.1017/erm.2023.27] [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: 04/26/2023] [Revised: 08/17/2023] [Accepted: 12/05/2023] [Indexed: 04/04/2024]
Abstract
Manipulation of T cells has revolutionized cancer immunotherapy. Notably, the use of T cells carrying engineered T cell receptors (TCR-T) offers a favourable therapeutic pathway, particularly in the treatment of solid tumours. However, major challenges such as limited clinical response efficacy, off-target effects and tumour immunosuppressive microenvironment have hindered the clinical translation of this approach. In this review, we mainly want to guide TCR-T investigators on several major issues they face in the treatment of solid tumours after obtaining specific TCR sequences: (1) whether we have to undergo affinity maturation or not, and what parameter we should use as a criterion for being more effective. (2) What modifications can be added to counteract the tumour inhibitory microenvironment to make our specific T cells to be more effective and what is the safety profile of such modifications? (3) What are the new forms and possibilities for TCR-T cell therapy in the future?
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Affiliation(s)
- Guoheng Mo
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xinyu Lu
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Sha Wu
- Department of Immunology/Guangdong Provincial Key Laboratory of Proteomics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, People's Republic of China
| | - Wei Zhu
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
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O’Neal J, Cooper ML, Ritchey JK, Gladney S, Niswonger J, González LS, Street E, Haas GJ, Carter A, Amayta PN, Gao F, Lee BH, Choi D, Berrien-Elliott M, Zhou A, Fehniger TA, Rettig MP, DiPersio JF. Anti-myeloma efficacy of CAR-iNKT is enhanced with a long-acting IL-7, rhIL-7-hyFc. Blood Adv 2023; 7:6009-6022. [PMID: 37399471 PMCID: PMC10582278 DOI: 10.1182/bloodadvances.2023010032] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 05/30/2023] [Accepted: 06/19/2023] [Indexed: 07/05/2023] Open
Abstract
Multiple myeloma (MM), a malignancy of mature plasma cells, remains incurable. B-cell maturation antigen (BCMA) is the lead protein target for chimeric antigen receptor (CAR) therapy because of its high expression in most MM, with limited expression in other cell types, resulting in favorable on-target, off tumor toxicity. The response rate to autologous BCMA CAR-T therapy is high; however, it is not curative and is associated with risks of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome. Outcomes in patients treated with BCMA CAR-T cells (CAR-Ts) may improve with allogeneic CAR T-cell therapy, which offer higher cell fitness and reduced time to treatment. However, to prevent the risk of graft-versus-host disease (GVHD), allogenic BCMA CAR-Ts require genetic deletion of the T-cell receptor (TCR), which has potential for unexpected functional or phenotype changes. Invariant natural killer T cells (iNKTs) have an invariant TCR that does not cause GVHD and, as a result, can be used in an allogeneic setting without the need for TCR gene editing. We demonstrate significant anti-myeloma activity of BCMA CAR-iNKTs in a xenograft mouse model of myeloma. We found that a long-acting interleukin-7 (IL-7), rhIL-7-hyFc, significantly prolonged survival and reduced tumor burden in BCMA CAR-iNKT-treated mice in both primary and re-challenge settings. Furthermore, in CRS in vitro assays, CAR-iNKTs induced less IL-6 than CAR-Ts, suggesting a reduced likelihood of CAR-iNKT therapy to induce CRS in patients. These data suggest that BCMA CAR-iNKTs are potentially a safer, effective alternative to BCMA CAR-Ts and that BCMA CAR-iNKT efficacy is further potentiated with rhIL-7-hyFc.
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Affiliation(s)
- Julie O’Neal
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
| | - Matthew L. Cooper
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Julie K. Ritchey
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Susan Gladney
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Jessica Niswonger
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - L. Sofía González
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Emily Street
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Gabriel J. Haas
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Alun Carter
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Parmeshwar N. Amayta
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
| | - Feng Gao
- Division of Public Health Sciences, Department of Surgery, Washington University School of Medicine, Saint Louis, MO
| | | | | | - Melissa Berrien-Elliott
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
| | - Alice Zhou
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
| | - Todd A. Fehniger
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
| | - Mike P. Rettig
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
| | - John F. DiPersio
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO
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Bauer J, Rajagopal N, Gupta P, Gupta P, Nixon AE, Kumar S. How can we discover developable antibody-based biotherapeutics? Front Mol Biosci 2023; 10:1221626. [PMID: 37609373 PMCID: PMC10441133 DOI: 10.3389/fmolb.2023.1221626] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Accepted: 07/10/2023] [Indexed: 08/24/2023] Open
Abstract
Antibody-based biotherapeutics have emerged as a successful class of pharmaceuticals despite significant challenges and risks to their discovery and development. This review discusses the most frequently encountered hurdles in the research and development (R&D) of antibody-based biotherapeutics and proposes a conceptual framework called biopharmaceutical informatics. Our vision advocates for the syncretic use of computation and experimentation at every stage of biologic drug discovery, considering developability (manufacturability, safety, efficacy, and pharmacology) of potential drug candidates from the earliest stages of the drug discovery phase. The computational advances in recent years allow for more precise formulation of disease concepts, rapid identification, and validation of targets suitable for therapeutic intervention and discovery of potential biotherapeutics that can agonize or antagonize them. Furthermore, computational methods for de novo and epitope-specific antibody design are increasingly being developed, opening novel computationally driven opportunities for biologic drug discovery. Here, we review the opportunities and limitations of emerging computational approaches for optimizing antigens to generate robust immune responses, in silico generation of antibody sequences, discovery of potential antibody binders through virtual screening, assessment of hits, identification of lead drug candidates and their affinity maturation, and optimization for developability. The adoption of biopharmaceutical informatics across all aspects of drug discovery and development cycles should help bring affordable and effective biotherapeutics to patients more quickly.
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Affiliation(s)
- Joschka Bauer
- Early Stage Pharmaceutical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach/Riss, Germany
- In Silico Team, Boehringer Ingelheim, Hannover, Germany
| | - Nandhini Rajagopal
- In Silico Team, Boehringer Ingelheim, Hannover, Germany
- Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, United States
| | - Priyanka Gupta
- In Silico Team, Boehringer Ingelheim, Hannover, Germany
- Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, United States
| | - Pankaj Gupta
- In Silico Team, Boehringer Ingelheim, Hannover, Germany
- Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, United States
| | - Andrew E. Nixon
- Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, United States
| | - Sandeep Kumar
- In Silico Team, Boehringer Ingelheim, Hannover, Germany
- Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, United States
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Frigerio B, Montermini M, Canevari S, Figini M. Role of antibody engineering in generation of derivatives starting from MOv19 MAb: 40 years of biological/therapeutic tools against folate receptor alfa. Antib Ther 2022; 5:301-310. [PMID: 36518225 PMCID: PMC9743174 DOI: 10.1093/abt/tbac026] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 09/13/2022] [Accepted: 10/15/2022] [Indexed: 05/18/2024] Open
Abstract
In the 1980s, we developed and characterized numerous murine monoclonal antibodies (MAbs) directed against human tumor-associated antigens. This mini review is focused on the generation of derivatives of an anti-folate receptor α (FRα) MAbs, named MOv19, exploiting the antibody-engineering progresses in the last 40 years. The FRα location on the luminal surface of proliferating epithelial cells, inaccessible to circulation, versus its over-expression in the entire surface of numerous carcinomas suggested a role for anti-FRα MAbs in the diagnosis and/or treatment of solid tumors. Presently, two MOv19 derivatives are in clinical trials: a chimeric resurfaced version in an antibody-drug conjugate format (SORAYA trial, 2022) and the murine scFv in a second generation chimeric antigen receptor, CAR-T (Phase Ia, 2021). MOv19 and its derivatives could be considered a relevant example that well-characterized anti-tumor murine Mabs and antibody engineering could be combined to generate useful therapeutic tools.
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Affiliation(s)
- Barbara Frigerio
- Biomarkers Unit, Department of Applied Research and Technical Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy
| | - Matilde Montermini
- Biomarkers Unit, Department of Applied Research and Technical Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy
| | - Silvana Canevari
- Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy
| | - Mariangela Figini
- Biomarkers Unit, Department of Applied Research and Technical Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan 20133, Italy
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Chouhan P, Singh S, Sharma V, Prajapati VK. Anti-IL-10 Antibody Humanization by SDR Grafting with Enhanced Affinity to Neutralize the Adverse Response of Interleukin-10. Int J Pept Res Ther 2022. [DOI: 10.1007/s10989-022-10456-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
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11
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O'Neal J, Ritchey JK, Cooper ML, Niswonger J, Sofía González L, Street E, Rettig MP, Gladney SW, Gehrs L, Abboud R, Prior JL, Haas GJ, Jayasinghe RG, Ding L, Ghobadi A, Vij R, DiPersio JF. CS1 CAR-T targeting the distal domain of CS1 (SLAMF7) shows efficacy in high tumor burden myeloma model despite fratricide of CD8+CS1 expressing CAR-T cells. Leukemia 2022; 36:1625-1634. [PMID: 35422095 PMCID: PMC9162922 DOI: 10.1038/s41375-022-01559-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 03/11/2022] [Accepted: 03/24/2022] [Indexed: 11/09/2022]
Abstract
Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains - the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.
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Affiliation(s)
- Julie O'Neal
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA.
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA.
| | - Julie K Ritchey
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Matthew L Cooper
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
| | - Jessica Niswonger
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - L Sofía González
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Emily Street
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Michael P Rettig
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
| | - Susan W Gladney
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Leah Gehrs
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Ramzi Abboud
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
| | - Julie L Prior
- Department of Radiology, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Gabriel J Haas
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Reyka G Jayasinghe
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, 63108, USA
| | - Li Ding
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
- McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, 63108, USA
- Department of Genetics, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
| | - Armin Ghobadi
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
| | - Ravi Vij
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA
| | - John F DiPersio
- Department of Medicine, Washington University in Saint Louis, Saint Louis, MO, 63110, USA.
- Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA.
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12
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Brinth AR, Svenson K, Mosyak L, Cunningham O, Hickling T, Lambert M. Crystal structure of ultra-humanized anti-pTau Fab reveals how germline substitutions humanize CDRs without loss of binding'. Sci Rep 2022; 12:8699. [PMID: 35610505 PMCID: PMC9130293 DOI: 10.1038/s41598-022-12838-6] [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: 07/25/2021] [Accepted: 05/10/2022] [Indexed: 11/08/2022] Open
Abstract
Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology "Augmented Binary Substitution" (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.
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Affiliation(s)
- Alette R Brinth
- BioMedicine Design, Pfizer Worldwide R&D, Dublin, D22 V8F8, Ireland
| | - Kristine Svenson
- BioMedicine Design, Pfizer Worldwide R&D, Cambridge, MA, 02139, USA
| | - Lidia Mosyak
- BioMedicine Design, Pfizer Worldwide R&D, Cambridge, MA, 02139, USA
| | - Orla Cunningham
- Ultrahuman Ltd. Kreston Reeves LLP Innovation Hs, Ramsgate Rd, Sandwich, CT13 9FF, UK
| | - Timothy Hickling
- BioMedicine Design, Pfizer Worldwide R&D, Andover, MA, 01810, USA
| | - Matthew Lambert
- BioMedicine Design, Pfizer Worldwide R&D, Dublin, D22 V8F8, Ireland.
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13
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Khetan R, Curtis R, Deane CM, Hadsund JT, Kar U, Krawczyk K, Kuroda D, Robinson SA, Sormanni P, Tsumoto K, Warwicker J, Martin ACR. Current advances in biopharmaceutical informatics: guidelines, impact and challenges in the computational developability assessment of antibody therapeutics. MAbs 2022; 14:2020082. [PMID: 35104168 PMCID: PMC8812776 DOI: 10.1080/19420862.2021.2020082] [Citation(s) in RCA: 48] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Therapeutic monoclonal antibodies and their derivatives are key components of clinical pipelines in the global biopharmaceutical industry. The availability of large datasets of antibody sequences, structures, and biophysical properties is increasingly enabling the development of predictive models and computational tools for the "developability assessment" of antibody drug candidates. Here, we provide an overview of the antibody informatics tools applicable to the prediction of developability issues such as stability, aggregation, immunogenicity, and chemical degradation. We further evaluate the opportunities and challenges of using biopharmaceutical informatics for drug discovery and optimization. Finally, we discuss the potential of developability guidelines based on in silico metrics that can be used for the assessment of antibody stability and manufacturability.
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Affiliation(s)
- Rahul Khetan
- Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
| | - Robin Curtis
- Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
| | | | | | - Uddipan Kar
- Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
| | | | - Daisuke Kuroda
- Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan.,Medical Device Development and Regulation Research Center, School of Engineering, The University of Tokyo, Tokyo, Japan.,Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan
| | | | - Pietro Sormanni
- Chemistry of Health, Yusuf Hamied Department of Chemistry, University of Cambridge
| | - Kouhei Tsumoto
- Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan.,Medical Device Development and Regulation Research Center, School of Engineering, The University of Tokyo, Tokyo, Japan.,Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.,The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Jim Warwicker
- Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
| | - Andrew C R Martin
- Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK
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14
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Van Lent J, Breukers J, Ven K, Ampofo L, Horta S, Pollet F, Imbrechts M, Geukens N, Vanhoorelbeke K, Declerck P, Lammertyn J. Miniaturized single-cell technologies for monoclonal antibody discovery. LAB ON A CHIP 2021; 21:3627-3654. [PMID: 34505611 DOI: 10.1039/d1lc00243k] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery.
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Affiliation(s)
- Julie Van Lent
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
| | - Jolien Breukers
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
| | - Karen Ven
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
| | - Louanne Ampofo
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
- Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, Leuven 3000, Belgium
| | - Sara Horta
- Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk 8500, Belgium
| | - Francesca Pollet
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
| | - Maya Imbrechts
- Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, Leuven 3000, Belgium
- PharmAbs, The KU Leuven Antibody Center, KU Leuven, Leuven 3000, Belgium
| | - Nick Geukens
- PharmAbs, The KU Leuven Antibody Center, KU Leuven, Leuven 3000, Belgium
| | - Karen Vanhoorelbeke
- Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk 8500, Belgium
- PharmAbs, The KU Leuven Antibody Center, KU Leuven, Leuven 3000, Belgium
| | - Paul Declerck
- Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, Leuven 3000, Belgium
- PharmAbs, The KU Leuven Antibody Center, KU Leuven, Leuven 3000, Belgium
| | - Jeroen Lammertyn
- Department of Biosystems, Biosensors Group, KU Leuven, Leuven 3001, Belgium.
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15
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Fernández-Quintero ML, Georges G, Varga JM, Liedl KR. Ensembles in solution as a new paradigm for antibody structure prediction and design. MAbs 2021; 13:1923122. [PMID: 34030577 PMCID: PMC8158028 DOI: 10.1080/19420862.2021.1923122] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
The rise of antibodies as a promising and rapidly growing class of biotherapeutic proteins has motivated numerous studies to characterize and understand antibody structures. In the past decades, the number of antibody crystal structures increased substantially, which revolutionized the atomistic understanding of antibody functions. Even though numerous static structures are known, various biophysical properties of antibodies (i.e., specificity, hydrophobicity and stability) are governed by their dynamic character. Additionally, the importance of high-quality structures in structure–function relationship studies has substantially increased. These structure–function relationship studies have also created a demand for precise homology models of antibody structures, which allow rational antibody design and engineering when no crystal structure is available. Here, we discuss various aspects and challenges in antibody design and extend the paradigm of describing antibodies with only a single static structure to characterizing them as dynamic ensembles in solution.
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Affiliation(s)
- Monica L Fernández-Quintero
- Department of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria
| | - Guy Georges
- Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
| | - Janos M Varga
- Department of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria
| | - Klaus R Liedl
- Department of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria
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16
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Rossotti MA, Bélanger K, Henry KA, Tanha J. Immunogenicity and humanization of single‐domain antibodies. FEBS J 2021; 289:4304-4327. [DOI: 10.1111/febs.15809] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 02/26/2021] [Accepted: 03/08/2021] [Indexed: 12/12/2022]
Affiliation(s)
- Martin A. Rossotti
- Life Sciences Division Human Health Therapeutics Research Centre National Research Council Canada Ottawa Canada
| | - Kasandra Bélanger
- Life Sciences Division Human Health Therapeutics Research Centre National Research Council Canada Ottawa Canada
| | - Kevin A. Henry
- Life Sciences Division Human Health Therapeutics Research Centre National Research Council Canada Ottawa Canada
- Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa Canada
| | - Jamshid Tanha
- Life Sciences Division Human Health Therapeutics Research Centre National Research Council Canada Ottawa Canada
- Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa Canada
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17
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Ulitzka M, Carrara S, Grzeschik J, Kornmann H, Hock B, Kolmar H. Engineering therapeutic antibodies for patient safety: tackling the immunogenicity problem. Protein Eng Des Sel 2021; 33:5944198. [PMID: 33128053 DOI: 10.1093/protein/gzaa025] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2020] [Revised: 09/17/2020] [Accepted: 09/18/2020] [Indexed: 12/21/2022] Open
Abstract
Established monoclonal antibodies (mAbs) allow treatment of cancers, autoimmune diseases and other severe illnesses. Side effects either arise due to interaction with the target protein and its biology or result from of the patient's immune system reacting to the foreign protein. This immunogenic reaction against therapeutic antibodies is dependent on various factors. The presence of non-human sequences can trigger immune responses as well as chemical and post-translational modifications of the antibody. However, even fully human antibodies can induce immune response through T cell epitopes or aggregates. In this review, we briefly describe, how therapeutic antibodies can interact with the patient's immune system and summarize recent advancements in protein engineering and in silico methods to reduce immunogenicity of therapeutic monoclonal antibodies.
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Affiliation(s)
- Michael Ulitzka
- Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.,Ferring Darmstadt Labs, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany
| | - Stefania Carrara
- Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany.,Ferring Darmstadt Labs, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany
| | - Julius Grzeschik
- Ferring Darmstadt Labs, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany
| | - Henri Kornmann
- Ferring International Center S.A., Chemin de la Vergognausaz 50, CH-1162 Saint-Prex, Switzerland
| | - Björn Hock
- Ferring International Center S.A., Chemin de la Vergognausaz 50, CH-1162 Saint-Prex, Switzerland
| | - Harald Kolmar
- Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287 Darmstadt, Germany
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18
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Norman RA, Ambrosetti F, Bonvin AMJJ, Colwell LJ, Kelm S, Kumar S, Krawczyk K. Computational approaches to therapeutic antibody design: established methods and emerging trends. Brief Bioinform 2020; 21:1549-1567. [PMID: 31626279 PMCID: PMC7947987 DOI: 10.1093/bib/bbz095] [Citation(s) in RCA: 132] [Impact Index Per Article: 26.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Revised: 06/07/2019] [Accepted: 07/05/2019] [Indexed: 12/31/2022] Open
Abstract
Antibodies are proteins that recognize the molecular surfaces of potentially noxious molecules to mount an adaptive immune response or, in the case of autoimmune diseases, molecules that are part of healthy cells and tissues. Due to their binding versatility, antibodies are currently the largest class of biotherapeutics, with five monoclonal antibodies ranked in the top 10 blockbuster drugs. Computational advances in protein modelling and design can have a tangible impact on antibody-based therapeutic development. Antibody-specific computational protocols currently benefit from an increasing volume of data provided by next generation sequencing and application to related drug modalities based on traditional antibodies, such as nanobodies. Here we present a structured overview of available databases, methods and emerging trends in computational antibody analysis and contextualize them towards the engineering of candidate antibody therapeutics.
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19
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Nicolazzi C, Caron A, Tellier A, Trombe M, Pinkas J, Payne G, Carrez C, Guérif S, Maguin M, Baffa R, Fassan M, Adam J, Mangatal-Wade L, Blanc V. An Antibody-Drug Conjugate Targeting MUC1-Associated Carbohydrate CA6 Shows Promising Antitumor Activities. Mol Cancer Ther 2020; 19:1660-1669. [PMID: 32451330 DOI: 10.1158/1535-7163.mct-19-0826] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2019] [Revised: 01/14/2020] [Accepted: 05/15/2020] [Indexed: 11/16/2022]
Abstract
Glycosylation is a complex multienzyme-related process that is frequently deregulated in cancer. Aberrant glycosylation can lead to the generation of novel tumor surface-specific glycotopes that can be targeted by antibodies. Murine DS6 mAb (muDS6) was generated from serous ovary adenocarcinoma immunization. It recognizes CA6, a Mucin-1 (MUC1)-associated sialoglycotope that is highly detected in breast, ovarian, lung, and bladder carcinomas. SAR566658 antibody-drug conjugate (ADC) is a humanized DS6 (huDS6) antibody conjugated through a cleavable linker to the cytotoxic maytansinoid derivative drug, DM4. SAR566658 binds to tumor cells with subnanomolar affinity, allowing good ADC internalization and intracellular delivery of DM4, resulting in tumor cell death (IC50 from 1 to 7.3 nmol/L). SAR566658 showed in vivo antitumor efficacy against CA6-positive human pancreas, cervix, bladder, and ovary tumor xenografts and against three breast patient-derived xenografts. Tumor regression was observed in all tumor models with minimal effective dose correlating with CA6 expression. SAR566658 displayed better efficacy than standard-of-care nontargeted tubulin binders. These data support the development of SAR566658 in patients with CA6-expressing tumors.
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MESH Headings
- Animals
- Antibodies, Monoclonal/chemistry
- Antibodies, Monoclonal/therapeutic use
- Antibodies, Monoclonal, Humanized/chemistry
- Antibodies, Monoclonal, Humanized/therapeutic use
- Antigens, Neoplasm/chemistry
- Antigens, Neoplasm/immunology
- Antineoplastic Agents/chemistry
- Antineoplastic Agents/therapeutic use
- Apoptosis
- Carbohydrates/chemistry
- Cell Proliferation
- Female
- Humans
- Immunoconjugates/chemistry
- Immunoconjugates/therapeutic use
- Mice
- Mice, Inbred NOD
- Mice, SCID
- Mucin-1/chemistry
- Mucin-1/immunology
- Neoplasms/drug therapy
- Neoplasms/metabolism
- Neoplasms/pathology
- Tumor Cells, Cultured
- Xenograft Model Antitumor Assays
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20
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Kuroda D, Tsumoto K. Engineering Stability, Viscosity, and Immunogenicity of Antibodies by Computational Design. J Pharm Sci 2020; 109:1631-1651. [DOI: 10.1016/j.xphs.2020.01.011] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 12/25/2019] [Accepted: 01/10/2020] [Indexed: 12/18/2022]
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21
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Fernández-Quintero ML, Heiss MC, Liedl KR. Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution. Protein Eng Des Sel 2019; 32:411-422. [PMID: 32129452 PMCID: PMC7098879 DOI: 10.1093/protein/gzaa004] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 01/15/2020] [Accepted: 01/27/2020] [Indexed: 01/11/2023] Open
Abstract
Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.
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Affiliation(s)
- Monica L Fernández-Quintero
- Institute of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria
| | - Martin C Heiss
- Institute of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria
| | - Klaus R Liedl
- Institute of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria
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22
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Petrucci MT, Vozella F. The Anti-CD38 Antibody Therapy in Multiple Myeloma. Cells 2019; 8:E1629. [PMID: 31842517 PMCID: PMC6952883 DOI: 10.3390/cells8121629] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 12/04/2019] [Accepted: 12/09/2019] [Indexed: 01/08/2023] Open
Abstract
Multiple myeloma (MM) is the second-most common hematologic malignancy after diffuse large B-cell lymphoma. Despite the improvement in response and survival rates following the introduction of novel therapies, only a few patients are cured, and the majority of MM patients experience several relapses and receive multiple lines of treatment. Currently, bortezomib and lenalidomide are the core component of treatment both at the time of diagnosis and at the relapse as well as the new proteasome inhibitors (PIs), such as carfilzomib and ixazomib, and the next-generation immunomodulatory drug, pomalidomide, are now available for patients in relapse. In addition, drugs with a different mechanism of action, such as the histone deacetylase inhibitor and the monoclonal antibodies (MoAb) targeting SLAMF7 or CD38, are a part of the anti-myeloma armamentarium and are very important for heavily pretreated or double refractory to a PI and IMiD patients. In this paper, we focus on the efficacy as well as toxicities of CD38 antibodies used both as a single agent and in combination as multiple myeloma treatment.
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Affiliation(s)
- Maria Teresa Petrucci
- Hematology, Department of Translational and Precision Medicine, Azienda Ospedaliera Policlinico Umberto I, Sapienza University of Rome, Via Benevento 6, 00161 Rome, Italy;
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23
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Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies (Basel) 2019; 8:antib8040055. [PMID: 31816964 PMCID: PMC6963682 DOI: 10.3390/antib8040055] [Citation(s) in RCA: 298] [Impact Index Per Article: 49.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 11/25/2019] [Accepted: 11/28/2019] [Indexed: 12/11/2022] Open
Abstract
Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.
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Affiliation(s)
- Mark L. Chiu
- Drug Product Development Science, Janssen Research & Development, LLC, Malvern, PA 19355, USA
- Correspondence:
| | - Dennis R. Goulet
- Department of Medicinal Chemistry, University of Washington, P.O. Box 357610, Seattle, WA 98195-7610, USA;
| | - Alexey Teplyakov
- Biologics Research, Janssen Research & Development, LLC, Spring House, PA 19477, USA; (A.T.); (G.L.G.)
| | - Gary L. Gilliland
- Biologics Research, Janssen Research & Development, LLC, Spring House, PA 19477, USA; (A.T.); (G.L.G.)
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24
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Mayrhofer P, Kunert R. Nomenclature of humanized mAbs: Early concepts, current challenges and future perspectives. Hum Antibodies 2019; 27:37-51. [PMID: 30103312 PMCID: PMC6294595 DOI: 10.3233/hab-180347] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Nomenclature of monoclonal antibodies traditionally followed a strict scheme indicating target and species information. Because of the rapid advances in this field, emphasized by approval of four humanized and six human antibodies in 2017, the International Nonproprietary Name of new antibodies was updated profoundly by removing the species substem completely. In this review we give an overview about what developments led to the preference of the scientific community towards human-like antibodies. We summarize the major updates in naming schemes that tried to classify antibodies according to their humanization technique or to the final primary sequence and how this led to the erroneous perception to indicate expected immunogenicity. Following the new 2017 nomenclature update, there will not be any information available about the species origin in the names of new antibodies, which emphasizes the need for providing additional supplemental information to the scientific community and develop tools to accurately estimate and control the safety of new monoclonal antibody molecules.
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Affiliation(s)
- Patrick Mayrhofer
- Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria
| | - Renate Kunert
- Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria
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25
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Polli JR, Engler FA, Balthasar JP. Physiologically Based Modeling of the Pharmacokinetics of "Catch-and-Release" Anti-Carcinoembryonic Antigen Monoclonal Antibodies in Colorectal Cancer Xenograft Mouse Models. J Pharm Sci 2018; 108:674-691. [PMID: 30321546 DOI: 10.1016/j.xphs.2018.09.037] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Revised: 09/21/2018] [Accepted: 09/24/2018] [Indexed: 10/28/2022]
Abstract
Engineered monoclonal antibodies (mAbs) with pH-sensitive target release, or "catch-and-release" (CAR) binding, have shown promise in decreasing the extent of target-mediated mAb elimination, increasing mAb exposure, and increasing dose potency. This study developed a mechanistic physiologically based pharmacokinetic (PBPK) model to evaluate the effects of pH-sensitive CAR target binding on the disposition of anti-carcinoembryonic antigen (CEA) mAbs in mouse models of colorectal cancer. The PBPK model was qualified by comparing model-predicted plasma concentration-time data with data observed in tumor-bearing mice following the administration of T84.66, a "standard" anti-CEA mAb that demonstrates strong binding at pH 7.4 and 5.5. Further simulations evaluated the effects CAR pH-dependent binding, with decreasing CEA affinity with decreasing pH, on anti-CEA mAb plasma pharmacokinetics. Simulated data were compared with data observed for a novel CAR mAb, 10H6. The PBPK model provided precise parameter estimates, and excellent data characterization (median prediction error 18.4%) following fitting to T84.66 data. Simulations well predicted 10H6 data (median prediction error 21.4%). Sensitivity analyses demonstrated that key determinants of the disposition of CAR mAbs include the following: antigen binding affinity, the rate constant of mAb-CEA dissociation in acidified endosomes, antigen concentration, and the tumor vasculature reflection coefficient.
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Affiliation(s)
- Joseph Ryan Polli
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, New York 14215
| | - Frank A Engler
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, New York 14215
| | - Joseph P Balthasar
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, New York 14215.
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26
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Chaisri U, Chaicumpa W. Evolution of Therapeutic Antibodies, Influenza Virus Biology, Influenza, and Influenza Immunotherapy. BIOMED RESEARCH INTERNATIONAL 2018; 2018:9747549. [PMID: 29998138 PMCID: PMC5994580 DOI: 10.1155/2018/9747549] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/25/2017] [Revised: 03/19/2018] [Accepted: 03/31/2018] [Indexed: 02/07/2023]
Abstract
This narrative review article summarizes past and current technologies for generating antibodies for passive immunization/immunotherapy. Contemporary DNA and protein technologies have facilitated the development of engineered therapeutic monoclonal antibodies in a variety of formats according to the required effector functions. Chimeric, humanized, and human monoclonal antibodies to antigenic/epitopic myriads with less immunogenicity than animal-derived antibodies in human recipients can be produced in vitro. Immunotherapy with ready-to-use antibodies has gained wide acceptance as a powerful treatment against both infectious and noninfectious diseases. Influenza, a highly contagious disease, precipitates annual epidemics and occasional pandemics, resulting in high health and economic burden worldwide. Currently available drugs are becoming less and less effective against this rapidly mutating virus. Alternative treatment strategies are needed, particularly for individuals at high risk for severe morbidity. In a setting where vaccines are not yet protective or available, human antibodies that are broadly effective against various influenza subtypes could be highly efficacious in lowering morbidity and mortality and controlling unprecedented epidemic/pandemic. Prototypes of human single-chain antibodies to several conserved proteins of influenza virus with no Fc portion (hence, no ADE effect in recipients) are available. These antibodies have high potential as a novel, safe, and effective anti-influenza agent.
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Affiliation(s)
- Urai Chaisri
- Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
| | - Wanpen Chaicumpa
- Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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27
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Chen L, Tian Y, Zhan K, Chen A, Weng Z, Huang J, Li Y, Sun Y, Zheng H, Li Y. A humanized TCR retaining authentic specificity and affinity conferred potent anti-tumour cytotoxicity. Immunology 2018; 155:123-136. [PMID: 29645087 DOI: 10.1111/imm.12935] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Revised: 03/05/2018] [Accepted: 03/28/2018] [Indexed: 12/14/2022] Open
Abstract
The affinity of T-cell receptor (TCR) determines the efficacy of TCR-based immunotherapy. By using human leucocyte antigen (HLA)-A*02 transgenic mice, a TCR was generated previously specific for human tumour testis antigen peptide MAGE-A3112-120 (KVAELVHFL) HLA-A*02 complex. We developed an approach to humanize the murine TCR by replacing the mouse framework with sequences of folding optimized human TCR variable domains for retaining binding affinity. The resultant humanized TCR exhibited higher affinity and conferred better anti-tumour activity than its parent murine MAGE-A3 TCR (SRm1). In addition, the affinity of humanized TCR was enhanced further to achieve improved T-cell activation. Our studies demonstrated that the human TCR variable domain frameworks could provide support for complementarity-determining regions from a murine TCR, and retain the original binding activity. It could be used as a generic approach of TCR humanization.
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Affiliation(s)
- Lin Chen
- State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Ye Tian
- State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Kai Zhan
- XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd, Guangzhou, China
| | - Anan Chen
- State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Zhiming Weng
- XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd, Guangzhou, China
| | - Jiao Huang
- XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd, Guangzhou, China
| | - Yanyan Li
- School of Life Sciences, University of Science and Technology of China, Hefei, China
| | - Yongjie Sun
- Institute of Health Sciences, Anhui University, Hefei, China
| | - Hongjun Zheng
- XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd, Guangzhou, China
| | - Yi Li
- State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.,University of Chinese Academy of Sciences, Beijing, China.,XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd, Guangzhou, China
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28
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Khoo YL, Cheah SH, Chong H. Humanization of chimeric anti-CD20 antibody by logical and bioinformatics approach with retention of biological activity. Immunotherapy 2018; 9:567-577. [PMID: 28595518 DOI: 10.2217/imt-2017-0016] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
AIM To develop a fully bioactive humanized antibody from the chimeric rituximab for potential clinical applications using a relatively simpler and faster logical and bioinformatics approach. METHODS From bioinformatics data, mismatched mouse amino acids in variable light and heavy chain amphipathic regions were identified and substituted with those common to human antibody framework. Appropriate synthetic DNA sequences inserted into vectors were transfected into HEK293 cells to produce the humanized antibody. RESULTS Humanized antibodies showed specific binding to CD20 and greater cytotoxicity to cancer WIL2-NS cell proliferation than rituximab in vitro. CONCLUSION A humanized version of rituximab with potential to be developed into a biobetter for treatment of B-cell disorders has been successfully generated using a logical and bioinformatics approach.
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Affiliation(s)
- Yoke L Khoo
- Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.,Department of Quality Control, Inno Biologics Sdn. Bhd., Nilai, Negeri Sembilan, Malaysia
| | - Swee H Cheah
- Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Heilly Chong
- Department of Protein Science, Inno Biologics Sdn. Bhd., Nilai, Negeri Sembilan, Malaysia
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29
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Cooper ML, Choi J, Staser K, Ritchey JK, Devenport JM, Eckardt K, Rettig MP, Wang B, Eissenberg LG, Ghobadi A, Gehrs LN, Prior JL, Achilefu S, Miller CA, Fronick CC, O'Neal J, Gao F, Weinstock DM, Gutierrez A, Fulton RS, DiPersio JF. An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia 2018; 32:1970-1983. [PMID: 29483708 PMCID: PMC6102094 DOI: 10.1038/s41375-018-0065-5] [Citation(s) in RCA: 285] [Impact Index Per Article: 40.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Revised: 01/22/2018] [Accepted: 02/01/2018] [Indexed: 12/27/2022]
Abstract
T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here we describe a fratricide resistant ‘off-the-shelf’ CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide resistant, allo-tolerant ‘off-the-shelf’ CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma without a requirement for autologous T cells.
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Affiliation(s)
- Matthew L Cooper
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
| | - Jaebok Choi
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Karl Staser
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA.,Department of Internal Medicine, Division of Dermatology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Julie K Ritchey
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Jessica M Devenport
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Kayla Eckardt
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Michael P Rettig
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Bing Wang
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Linda G Eissenberg
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Armin Ghobadi
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Leah N Gehrs
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Julie L Prior
- Mallinckrodt Institute of Radiology, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110, USA
| | - Samuel Achilefu
- Mallinckrodt Institute of Radiology, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110, USA
| | - Christopher A Miller
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA.,McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, 63108, USA
| | - Catrina C Fronick
- McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, 63108, USA
| | - Julie O'Neal
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Feng Gao
- Department of Surgery, Division of Public Health Sciences, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - David M Weinstock
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
| | - Alejandro Gutierrez
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.,Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, 02215, USA
| | - Robert S Fulton
- McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, 63108, USA
| | - John F DiPersio
- Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
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30
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Abstract
Antibody humanization process converts any nonhuman antibody sequence into humanized antibodies. This can be achieved using different methods of antibody design and engineering. This chapter will primarily focus on antibody design using a homology model followed by framework shuffling of murine to human germline sequence for humanization. Historically, mouse antibodies have been humanized using sequence-based approaches, in which all the murine frameworks are replaced with most homologous human germline sequence or related scaffold. Most often this humanized antibody design, when tested, has a significantly reduced binding or no binding to the cognate antigen. This is due to noncompatibility of mouse CDRs being supported by non-native human framework scaffold. This mismatch between mouse, human structural fold, and elimination of key conformational residues often leads to antibody humanization failures. Recently, there has been advent of homology modelor structure-guided antibody humanization. Instead of humanization based on linear sequence, this approach takes into account the tertiary structure and fold of the mouse antibody. A mouse homology model of the fragment variable is created, and based on sequence alignment with human germline, residues that are different in mouse are replaced with humanized sequence in the model. Energy minimization is applied to this humanized model that also delineates residues which might have steric clashes due to change in the overall tertiary conformation of the humanized antibody. Therefore, a homology model-guided with rational mutations, and reintroduction of key conformational residues from mouse antibody not only eliminates steric clashes but might also restore function in relation to binding affinity to its antigen.
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Affiliation(s)
- Vinodh B Kurella
- Protein Engineering, Immuno-Oncology Division, Intrexon Corporation, Germantown, MD, USA
- Merrimack Pharmaceuticals, Protein Engineering, Cambridge, MA, USA
| | - Reddy Gali
- The Harvard Clinical and Translational Science Center and Countway Library of Medicine, Harvard Medical School, Boston, MA, USA.
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.
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31
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Qiu J, Qiu T, Huang Y, Cao Z. Identifying the Epitope Regions of Therapeutic Antibodies Based on Structure Descriptors. Int J Mol Sci 2017; 18:E2457. [PMID: 29186775 PMCID: PMC5751102 DOI: 10.3390/ijms18122457] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Revised: 11/13/2017] [Accepted: 11/13/2017] [Indexed: 11/16/2022] Open
Abstract
Therapeutic antibodies are widely used for disease detection and specific treatments. However, as an exogenous protein, these antibodies can be detected by the human immune system and elicit a response that can lead to serious illnesses. Therapeutic antibodies can be engineered through antibody humanization, which aims to maintain the specificity and biological function of the original antibodies, and reduce immunogenicity. However, the antibody drug effect is synchronously reduced as more exogenous parts are replaced by human antibodies. Hence, a major challenge in this area is to precisely detect the epitope regions in immunogenic antibodies and guide point mutations of exogenous antibodies to balance both humanization level and drug effect. In this article, the latest dataset of immunoglobulin complexes was collected from protein data bank (PDB) to discover the spatial features of immunogenic antibody. Furthermore, a series of structure descriptors were generated to characterize and distinguish epitope residues from non-immunogenic regions. Finally, a computational model was established based on structure descriptors, and results indicated that this model has the potential to precisely predict the epitope regions of therapeutic antibodies. With rapid accumulation of immunoglobulin complexes, this methodology could be used to improve and guide future antibody humanization and potential clinical applications.
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Affiliation(s)
- Jingxuan Qiu
- School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; (J.Q.); (Y.H.)
- School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Tianyi Qiu
- The Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China;
| | - Yin Huang
- School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; (J.Q.); (Y.H.)
| | - Zhiwei Cao
- School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; (J.Q.); (Y.H.)
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32
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Parola C, Neumeier D, Reddy ST. Integrating high-throughput screening and sequencing for monoclonal antibody discovery and engineering. Immunology 2017; 153:31-41. [PMID: 28898398 DOI: 10.1111/imm.12838] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2017] [Revised: 09/06/2017] [Accepted: 09/06/2017] [Indexed: 12/14/2022] Open
Abstract
Monoclonal antibody discovery and engineering is a field that has traditionally been dominated by high-throughput screening platforms (e.g. hybridomas and surface display). In recent years the emergence of high-throughput sequencing has made it possible to obtain large-scale information on antibody repertoire diversity. Additionally, it has now become more routine to perform high-throughput sequencing on antibody repertoires to also directly discover antibodies. In this review, we provide an overview of the progress in this field to date and show how high-throughput screening and sequencing are converging to deliver powerful new workflows for monoclonal antibody discovery and engineering.
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Affiliation(s)
- Cristina Parola
- Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.,Life Science Zurich Graduate School, Systems Biology, ETH Zurich, University of Zurich, Zurich, Switzerland
| | - Daniel Neumeier
- Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland
| | - Sai T Reddy
- Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland
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33
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Markovsky E, Vax E, Ben-Shushan D, Eldar-Boock A, Shukrun R, Yeini E, Barshack I, Caspi R, Harari-Steinberg O, Pode-Shakked N, Dekel B, Satchi-Fainaro R. Wilms Tumor NCAM-Expressing Cancer Stem Cells as Potential Therapeutic Target for Polymeric Nanomedicine. Mol Cancer Ther 2017; 16:2462-2472. [DOI: 10.1158/1535-7163.mct-17-0184] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 06/14/2017] [Accepted: 07/13/2017] [Indexed: 11/16/2022]
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34
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Loebrich S, Shen M, Cohen E, Payne G, Chen Y, Bogalhas M, Zhao Y. Development and Characterization of a Neutralizing Anti-idiotype Antibody Against Mirvetuximab for Analysis of Clinical Samples. AAPS JOURNAL 2017; 19:1223-1234. [DOI: 10.1208/s12248-017-0098-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/09/2017] [Accepted: 05/04/2017] [Indexed: 11/30/2022]
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35
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A phase 1b study of isatuximab plus lenalidomide and dexamethasone for relapsed/refractory multiple myeloma. Blood 2017; 129:3294-3303. [PMID: 28483761 DOI: 10.1182/blood-2016-09-740787] [Citation(s) in RCA: 140] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2016] [Accepted: 03/21/2017] [Indexed: 12/11/2022] Open
Abstract
This phase 1b, open-label, dose-escalation study assessed the safety, efficacy, and pharmacokinetics of anti-CD38 monoclonal antibody isatuximab given in 2 schedules (3, 5, or 10 mg/kg every other week [Q2W] or 10 or 20 mg/kg weekly [QW] for 4 weeks and then Q2W thereafter [QW/Q2W]), in combination with lenalidomide 25 mg (days 1-21) and dexamethasone 40 mg (QW), in patients with relapsed/refractory multiple myeloma (RRMM). Patients received 28-day treatment cycles; the primary objective was to determine the maximum tolerated dose (MTD) of isatuximab with lenalidomide and dexamethasone. Fifty-seven patients (median 5 [range 1-12] prior regimens; 83% refractory to previous lenalidomide therapy) were treated. Median duration of dosing was 36.4 weeks; 15 patients remained on treatment at data cutoff. Isatuximab-lenalidomide-dexamethasone was generally well tolerated with only 1 dose-limiting toxicity reported (grade 3 pneumonia at 20 mg/kg QW/Q2W); the MTD was not reached. The most common isatuximab-related adverse events were infusion-associated reactions (IARs) (56%), which were grade 1/2 in 84% of patients who had an IAR and predominantly occurred during the first infusion. In the efficacy-evaluable population, the overall response rate (ORR) was 56% (29/52) and was similar between the 10 mg/kg Q2W and 10 and 20 mg/kg QW/Q2W cohorts. The ORR was 52% in 42 evaluable lenalidomide-refractory patients. Overall median progression-free survival was 8.5 months. Isatuximab exposure increased in a greater than dose-proportional manner; isatuximab and lenalidomide pharmacokinetic parameters appeared independent. These data suggest that isatuximab combined with lenalidomide and dexamethasone is active and tolerated in heavily pretreated patients with RRMM. This trial was registered at www.clinicaltrials.gov as #NCT01749969.
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36
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Targeting NCAM-expressing neuroblastoma with polymeric precision nanomedicine. J Control Release 2017; 249:162-172. [DOI: 10.1016/j.jconrel.2017.01.044] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Revised: 01/24/2017] [Accepted: 01/30/2017] [Indexed: 01/01/2023]
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37
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Choi Y, Ndong C, Griswold KE, Bailey-Kellogg C. Computationally driven antibody engineering enables simultaneous humanization and thermostabilization. Protein Eng Des Sel 2016; 29:419-426. [PMID: 27334453 DOI: 10.1093/protein/gzw024] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2016] [Accepted: 05/25/2016] [Indexed: 12/22/2022] Open
Abstract
Humanization reduces the immunogenicity risk of therapeutic antibodies of non-human origin. Thermostabilization can be critical for clinical development and application of therapeutic antibodies. Here, we show that the computational antibody redesign method Computationally Driven Antibody Humanization (CoDAH) enables these two goals to be accomplished simultaneously and seamlessly. A panel of CoDAH designs for the murine parent of cetuximab, a chimeric anti-EGFR antibody, exhibited both substantially improved thermostabilities and substantially higher levels of humanness, while retaining binding activity near the parental level. The consistently high quality of the turnkey CoDAH designs, over a whole panel of variants, suggests that the computationally directed approach encapsulates key determinants of antibody structure and function.
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Affiliation(s)
- Yoonjoo Choi
- Department of Computer Science, Dartmouth College, Hanover, NH 03755, USA
| | - Christian Ndong
- Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA
| | - Karl E Griswold
- Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA.,Norris Cotton Cancer Center at Dartmouth, Lebanon, NH 03766, USA.,Department of Biological Sciences, Dartmouth, Hanover, NH 03755, USA
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38
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Martin AC, Rees AR. Extracting human antibody sequences from public databases for antibody humanization: high frequency of species assignment errors. Protein Eng Des Sel 2016; 29:403-408. [DOI: 10.1093/protein/gzw018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Accepted: 05/09/2016] [Indexed: 11/13/2022] Open
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39
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Dehaini D, Fang RH, Zhang L. Biomimetic strategies for targeted nanoparticle delivery. Bioeng Transl Med 2016; 1:30-46. [PMID: 29313005 PMCID: PMC5689512 DOI: 10.1002/btm2.10004] [Citation(s) in RCA: 102] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2016] [Revised: 04/07/2016] [Accepted: 04/08/2016] [Indexed: 01/02/2023] Open
Abstract
Nanoparticle‐based drug delivery and imaging platforms have become increasingly popular over the past several decades. Among different design parameters that can affect their performance, the incorporation of targeting functionality onto nanoparticle surfaces has been a widely studied subject. Targeted formulations have the ability to improve efficacy and function by positively modulating tissue localization. Many methods exist for creating targeted nanoformulations, including the use of custom biomolecules such as antibodies or aptamers. More recently, a great amount of focus has been placed on biomimetic targeting strategies that leverage targeting interactions found directly in nature. Such strategies, which have been painstakingly selected over time by the process of evolution to maximize functionality, oftentimes enable scientists to forgo the specialized discovery processes associated with many traditional ligands and help to accelerate development of novel nanoparticle formulations. In this review, we categorize and discuss in‐depth recent works in this growing field of bioinspired research.
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Affiliation(s)
- Diana Dehaini
- Dept. of NanoEngineering and Moores Cancer Center University of California San Diego, La Jolla CA 92093
| | - Ronnie H Fang
- Dept. of NanoEngineering and Moores Cancer Center University of California San Diego, La Jolla CA 92093
| | - Liangfang Zhang
- Dept. of NanoEngineering and Moores Cancer Center University of California San Diego, La Jolla CA 92093
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40
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Kunert R, Reinhart D. Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 2016; 100:3451-61. [PMID: 26936774 PMCID: PMC4803805 DOI: 10.1007/s00253-016-7388-9] [Citation(s) in RCA: 271] [Impact Index Per Article: 30.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Revised: 02/07/2016] [Accepted: 02/09/2016] [Indexed: 01/16/2023]
Abstract
Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.
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Affiliation(s)
- Renate Kunert
- Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190, Vienna, Austria.
| | - David Reinhart
- Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190, Vienna, Austria
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Margreitter C, Mayrhofer P, Kunert R, Oostenbrink C. Antibody humanization by molecular dynamics simulations-in-silico guided selection of critical backmutations. J Mol Recognit 2016; 29:266-75. [PMID: 26748949 PMCID: PMC4948679 DOI: 10.1002/jmr.2527] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2015] [Revised: 11/19/2015] [Accepted: 11/20/2015] [Indexed: 11/09/2022]
Abstract
Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. "Superhumanization" describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody-antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.
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Affiliation(s)
- Christian Margreitter
- Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, 1190, Austria
| | - Patrick Mayrhofer
- Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, 1190, Austria
| | - Renate Kunert
- Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, 1190, Austria
| | - Chris Oostenbrink
- Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, 1190, Austria
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Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies. Proc Natl Acad Sci U S A 2015; 112:15354-9. [PMID: 26621728 DOI: 10.1073/pnas.1510944112] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.
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Zielonka S, Empting M, Grzeschik J, Könning D, Barelle CJ, Kolmar H. Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 2015; 7:15-25. [PMID: 25523873 PMCID: PMC4622739 DOI: 10.4161/19420862.2015.989032] [Citation(s) in RCA: 105] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.
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Key Words
- CDR, complementarity-determining region
- HV, hypervariable region
- IgNAR
- IgNAR V domain, variable domain of IgNAR
- IgNAR, immunoglobulin new antigen receptor
- VH, variable domain of the heavy chain
- VHH, variable domain of camelid heavy chain antibodies
- VL, variable domain of the light chain
- antibody technology
- biologic therapeutic
- heavy chain antibody
- mAbs, monoclonal antibodies
- scFv, single chain variable fragment
- shark
- single chain binding domain
- vNAR, variable domain of IgNAR
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Affiliation(s)
- Stefan Zielonka
- a Institute for Organic Chemistry and Biochemistry ; Technische Universität Darmstadt ; Darmstadt , Germany
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Choi Y, Hua C, Sentman CL, Ackerman ME, Bailey-Kellogg C. Antibody humanization by structure-based computational protein design. MAbs 2015; 7:1045-57. [PMID: 26252731 PMCID: PMC5045135 DOI: 10.1080/19420862.2015.1076600] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2015] [Revised: 07/06/2015] [Accepted: 07/20/2015] [Indexed: 12/15/2022] Open
Abstract
Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH ("Computationally-Driven Antibody Humanization") in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.
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Affiliation(s)
- Yoonjoo Choi
- Department of Computer Science; Dartmouth College; Hanover, NH USA
| | - Casey Hua
- Thayer School of Engineering; Dartmouth College; Hanover, NH USA
- Department of Microbiology and Immunology; Geisel School of Medicine; Dartmouth College; Lebanon, NH USA
| | - Charles L Sentman
- Department of Microbiology and Immunology; Geisel School of Medicine; Dartmouth College; Lebanon, NH USA
| | - Margaret E Ackerman
- Thayer School of Engineering; Dartmouth College; Hanover, NH USA
- Department of Microbiology and Immunology; Geisel School of Medicine; Dartmouth College; Lebanon, NH USA
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Hong EE, Erickson H, Lutz RJ, Whiteman KR, Jones G, Kovtun Y, Blanc V, Lambert JM. Design of Coltuximab Ravtansine, a CD19-Targeting Antibody-Drug Conjugate (ADC) for the Treatment of B-Cell Malignancies: Structure-Activity Relationships and Preclinical Evaluation. Mol Pharm 2015; 12:1703-16. [PMID: 25856201 DOI: 10.1021/acs.molpharmaceut.5b00175] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Coltuximab ravtansine (SAR3419) is an antibody-drug conjugate (ADC) targeting CD19 created by conjugating a derivative of the potent microtubule-acting cytotoxic agent, maytansine, to a version of the anti-CD19 antibody, anti-B4, that was humanized as an IgG1 by variable domain resurfacing. Four different linker-maytansinoid constructs were synthesized (average ∼3.5 maytansinoids/antibody for each) to evaluate the impact of linker-payload design on the activity of the maytansinoid-ADCs targeting CD19. The ADC composed of DM4 (N(2')-deacetyl-N(2')-[4-mercapto-4-methyl-1-oxopentyl]maytansine) conjugated to antibody via the N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker was selected for development as SAR3419. A molar ratio for DM4/antibody of between 3 and 5 was selected for the final design of SAR3419. Evaluation of SAR3419 in Ramos tumor xenograft models showed that the minimal effective single dose was about 50 μg/kg conjugated DM4 (∼2.5 mg/kg conjugated antibody), while twice this dose gave complete regressions in 100% of the mice. SAR3419 arrests cells in the G2/M phase of the cell cycle, ultimately leading to apoptosis after about 24 h. The results of in vitro and in vivo studies with SAR3419 made with DM4 that was [(3)H]-labeled at the C20 methoxy group of the maytansinoid suggest a mechanism of internalization and intracellular trafficking of SAR3419, ultimately to lysosomes, in which the antibody is fully degraded, releasing lysine-N(ε)-SPDB-DM4 as the initial metabolite. Subsequent intracellular reduction of the disulfide bond between linker and DM4 generates the free thiol species, which is then converted to S-methyl DM4 by cellular methyl transferase activity. We provide evidence to suggest that generation of S-methyl DM4 in tumor cells may contribute to in vivo tumor eradication via bystander killing of neighboring tumor cells. Furthermore, we show that S-methyl DM4 is converted to the sulfoxide and sulfone derivatives in the liver, suggesting that hepatic catabolism of the payload to less cytotoxic maytansinoid species contributes to the overall therapeutic window of SAR3419. This compound is currently in phase II clinical evaluation for the treatment of diffuse large B cell lymphoma.
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Affiliation(s)
- E Erica Hong
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Hans Erickson
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Robert J Lutz
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Kathleen R Whiteman
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Gregory Jones
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Yelena Kovtun
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
| | - Veronique Blanc
- §Sanofi, 13 quai Jules Guesde, Vitry sur Seine, 94403 France
| | - John M Lambert
- †ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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Ab O, Whiteman KR, Bartle LM, Sun X, Singh R, Tavares D, LaBelle A, Payne G, Lutz RJ, Pinkas J, Goldmacher VS, Chittenden T, Lambert JM. IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors. Mol Cancer Ther 2015; 14:1605-13. [PMID: 25904506 DOI: 10.1158/1535-7163.mct-14-1095] [Citation(s) in RCA: 162] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2014] [Accepted: 04/10/2015] [Indexed: 11/16/2022]
Abstract
A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors.
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Affiliation(s)
- Olga Ab
- Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts.
| | - Kathleen R Whiteman
- Department of Pharmacology Toxicology, ImmunoGen, Inc., Waltham, Massachusetts
| | - Laura M Bartle
- Department of Cell Biology, ImmunoGen, Inc., Waltham, Massachusetts
| | - Xiuxia Sun
- Department of Biochemistry, ImmunoGen, Inc., Waltham, Massachusetts
| | - Rajeeva Singh
- Department of Biochemistry, ImmunoGen, Inc., Waltham, Massachusetts
| | - Daniel Tavares
- Department of Antibody Engineering, ImmunoGen, Inc., Waltham, Massachusetts
| | - Alyssa LaBelle
- Department of Biomarkers, ImmunoGen, Inc., Waltham, Massachusetts
| | - Gillian Payne
- Department of Bioanalytical Science, ImmunoGen, Inc., Waltham, Massachusetts
| | - Robert J Lutz
- Department of Translational Research and Development, ImmunoGen, Inc., Waltham, Massachusetts
| | - Jan Pinkas
- Department of Pharmacology Toxicology, ImmunoGen, Inc., Waltham, Massachusetts
| | | | | | - John M Lambert
- Research and Development, ImmunoGen, Inc., Waltham, Massachusetts
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Deyev SM, Lebedenko EN, Petrovskaya LE, Dolgikh DA, Gabibov AG, Kirpichnikov MP. Man-made antibodies and immunoconjugates with desired properties: function optimization using structural engineering. RUSSIAN CHEMICAL REVIEWS 2015. [DOI: 10.1070/rcr4459] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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48
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Deckert J, Wetzel MC, Bartle LM, Skaletskaya A, Goldmacher VS, Vallée F, Zhou-Liu Q, Ferrari P, Pouzieux S, Lahoute C, Dumontet C, Plesa A, Chiron M, Lejeune P, Chittenden T, Park PU, Blanc V. SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+ Hematologic Malignancies. Clin Cancer Res 2014; 20:4574-83. [DOI: 10.1158/1078-0432.ccr-14-0695] [Citation(s) in RCA: 216] [Impact Index Per Article: 19.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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49
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A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies. Blood 2013; 122:3500-10. [PMID: 24002446 DOI: 10.1182/blood-2013-05-505685] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.
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50
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Lambert JM. Drug-conjugated antibodies for the treatment of cancer. Br J Clin Pharmacol 2013; 76:248-62. [PMID: 23173552 PMCID: PMC3731599 DOI: 10.1111/bcp.12044] [Citation(s) in RCA: 116] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2012] [Accepted: 10/25/2012] [Indexed: 12/27/2022] Open
Abstract
Despite considerable effort, application of monoclonal antibody technology has had only modest success in improving treatment outcomes in patients with solid tumours. Enhancing the cancer cell-killing activity of antibodies through conjugation to highly potent cytotoxic 'payloads' to create antibody-drug conjuates (ADCs) offers a strategy for developing anti-cancer drugs of great promise. Early ADCs exhibited side-effect profiles similar to those of 'classical' chemotherapeutic agents and their performance in clinical trials in cancer patients was generally poor. However, the recent clinical development of ADCs that have highly potent tubulin-acting agents as their payloads have profoundly changed the outlook for ADC technology. Twenty-five such ADCs are in clinical development and one, brentuximab vedotin, was approved by the FDA in August, 2011, for the treatment of patients with Hodgkin's lymphoma and patients with anaplastic large cell lymphoma, based on a high rate of durable responses in single arm phase II clinical trials. More recently, a second ADC, trastuzumab emtansine, has shown excellent anti-tumour activity with the presentation of results of a 991-patient randomized phase III trial in patients with HER2-positive metastatic breast cancer. Treatment with this ADC (single agent) resulted in a significantly improved progression-free survival of 9.6 months compared with 6.4 months for lapatinib plus capecitabine in the comparator arm and significantly prolonged overall survival. Besides demonstrating excellent efficacy, these ADCs were remarkably well tolerated. Thus these, and other ADCs in development, promise to achieve the long sought goal of ADC technology, that is, of having compounds with high anti-tumour activity at doses where adverse effects are generally mild.
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Affiliation(s)
- John M Lambert
- ImmunoGen, Inc, 830 Winter Street, Waltham, MA 02451, USA.
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