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1
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Glanville J, Bellin M, Pletnev S, Zhang B, Andrade JC, Kim S, Tsao D, Verardi R, Bedi R, Liao S, Newland R, Bayless NL, Youssef S, Tully ES, Bylund T, Kim S, Hirou H, Liu T, Kwong PD. Snake venom protection by a cocktail of varespladib and broadly neutralizing human antibodies. Cell 2025; 188:3117-3134.e11. [PMID: 40318633 DOI: 10.1016/j.cell.2025.03.050] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 12/18/2024] [Accepted: 03/31/2025] [Indexed: 05/07/2025]
Abstract
Snake envenomation is a neglected tropical disease, with 600 species causing over 100,000 deaths and 300,000 permanent disabilities in humans annually. Broadly neutralizing antibodies and broad chemical inhibitors have been proposed as solutions, but how to develop a therapeutically effective cocktail and the number of required components have been unclear. To address this gap, we iteratively recovered two broadly neutralizing antivenom antibodies from the memory B cells of a hyperimmune human donor with extensive snake venom exposure. The antibodies recognized conserved neutralizing epitopes on prevalent long and short snake neurotoxins, with crystal structures revealing antibody mimicry of the interfaces between these neurotoxins and their host target, the nicotinic acetylcholine receptor. We combined and tested these antibodies and the phospholipase inhibitor varespladib. A 3-component cocktail rescued animals from whole-venom challenge of all species in a 19-member WHO Category 1 and Category 2 elapid diversity set, with complete protection against most snakes observed.
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Affiliation(s)
- Jacob Glanville
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA.
| | - Mark Bellin
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Sergei Pletnev
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Baoshan Zhang
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | | | - Sangil Kim
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - David Tsao
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Raffaello Verardi
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Rishi Bedi
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Sindy Liao
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Raymond Newland
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Nicholas L Bayless
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Sawsan Youssef
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Ena S Tully
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Tatsiana Bylund
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Sujeong Kim
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Hannah Hirou
- Centivax, Inc., 1 Tower Place, Suite 800, South San Francisco, CA 94080, USA
| | - Tracy Liu
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Peter D Kwong
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, and Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
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2
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Beaulaurier J, Ly L, Duty JA, Tyer C, Stevens C, Hung CT, Sookdeo A, Drong AW, Kowdle S, Guzman-Solis A, Tortorella D, Turner DJ, Juul S, Hickey S, Lee B. De novo antibody identification in human blood from full-length single B cell transcriptomics and matching haplotype-resolved germline assemblies. Genome Res 2025; 35:929-941. [PMID: 40118521 PMCID: PMC12047243 DOI: 10.1101/gr.279392.124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Accepted: 02/12/2025] [Indexed: 03/23/2025]
Abstract
Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells results in whole transcriptome characterization of each cell, as well as highly accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrate binding to measles antigens and neutralization of authentic measles virus.
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Affiliation(s)
- John Beaulaurier
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Lynn Ly
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - J Andrew Duty
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Carly Tyer
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Christian Stevens
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Chuan-Tien Hung
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Akash Sookdeo
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Alex W Drong
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Shreyas Kowdle
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | - Axel Guzman-Solis
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
| | | | - Daniel J Turner
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Sissel Juul
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA
| | - Scott Hickey
- Oxford Nanopore Technologies, Inc., New York, New York 10013, USA;
| | - Benhur Lee
- Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA
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3
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Xu X, Rigas YE, Mattapallil MJ, Guo J, Nagarajan V, Bohrnsen E, Richards C, Gupta A, Gaud G, Love PE, Jiang T, Zhang A, Xu B, Peng Z, Jittayasothorn Y, Carr M, Magone MT, Brandes NT, Shane J, Schwarz B, St. Leger AJ, Caspi RR. Commensal-derived Trehalose Monocorynomycolate Triggers γδ T Cell-driven Protective Ocular Barrier Immunity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.17.643820. [PMID: 40166223 PMCID: PMC11956969 DOI: 10.1101/2025.03.17.643820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identify trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis (C. mast) as a potent stimulator of IL-17 production by γδ T cells at the ocular surface. Mechanistically, TMCM-driven IL-17 responses require both IL-1 signals and γδ TCR signaling, which also supports endogenous γδ T cell IL-1R1 expression. Notably, synthetic TMCM alone is sufficient to mimic the effect of C. mast in inducing γδ T cell immunity and protect against pathogenic corneal infection. Our findings establish TMCM as a key mediator of commensal-driven immune defense, highlighting its potential as a γδ T cell adjuvant and a microbiome-informed therapeutic to enhance IL-17-driven protection at barrier sites such as the ocular surface.
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Affiliation(s)
- Xiaoyan Xu
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | - Yannis E. Rigas
- Univ of Pittsburgh Sch Med, Dept Ophthalmol & Immunol, Pittsburgh, PA 15213, USA
| | | | - Jing Guo
- Dept Microbiol & Immunol, Stanford Univ Sch Med, Stanford, CA 94305, USA
| | | | - Eric Bohrnsen
- Res & Tech Br, Rocky Mountain Labs, NIAID, NIH, Hamilton, MT, USA
| | - Crystal Richards
- Res & Tech Br, Rocky Mountain Labs, NIAID, NIH, Hamilton, MT, USA
- Current address: Health Research & Development Branch, NIGMS, NIH
| | - Akriti Gupta
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | - Guillaume Gaud
- Hematopoiesis & Lymphocyte Biol Sctn, NICHD, NIH, Bethesda, MD, USA
| | - Paul E. Love
- Hematopoiesis & Lymphocyte Biol Sctn, NICHD, NIH, Bethesda, MD, USA
| | - Timothy Jiang
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | - Amy Zhang
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | - Biying Xu
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | - Zixuan Peng
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
| | | | - Mary Carr
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
- Dept Cell & Mol Biol, Univ Mississippi Med Center, Jackson, MS 39216, Current address: Dept Microbiol & Immunol, Emory Univ Sch Med, Atlanta, GA 30322, USA
| | | | | | - Jackie Shane
- Univ of Pittsburgh Sch Med, Dept Ophthalmol & Immunol, Pittsburgh, PA 15213, USA
| | - Benjamin Schwarz
- Res & Tech Br, Rocky Mountain Labs, NIAID, NIH, Hamilton, MT, USA
| | - Anthony J. St. Leger
- Univ of Pittsburgh Sch Med, Dept Ophthalmol & Immunol, Pittsburgh, PA 15213, USA
| | - Rachel R. Caspi
- Immunoreg Sctn, Lab Immunol, NEI, NIH, Bethesda MD 20892, USA
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4
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Kannan D, Wang E, Deeks SG, Lewin SR, Chakraborty AK. Mechanism for evolution of diverse autologous antibodies upon broadly neutralizing antibody therapy of people with HIV. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.05.641732. [PMID: 40161612 PMCID: PMC11952291 DOI: 10.1101/2025.03.05.641732] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Antiretroviral therapy (ART) inhibits Human Immunodeficiency Virus (HIV) replication to maintain undetectable viral loads in people living with HIV, but does not result in a cure. Due to the significant challenges of lifelong ART for many, there is strong interest in therapeutic strategies that result in cure. Recent clinical trials have shown that administration of broadly neutralizing antibodies (bnAbs) when there is some viremia can lead to ART-free viral control in some people; however, the underlying mechanisms are unclear. Our computational modeling shows that bnAbs administered in the presence of some viremia promote the evolution of autologous antibodies (aAbs) that target diverse epitopes of HIV spike proteins. This "net" of polyclonal aAbs could confer control since evasion of this response would require developing mutations in multiple epitopes. Our results provide a common mechanistic framework underlying recent clinical observations upon bnAb/ART therapy, and they should also motivate and inform new trials.
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Affiliation(s)
- Deepti Kannan
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Eric Wang
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Steven G. Deeks
- Department of Medicine, University of California, San Francisco, USA
| | - Sharon R. Lewin
- Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
- Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
- Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Australia
| | - Arup K. Chakraborty
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02139, USA
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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5
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Czarnecka M, Findik N, Schlör A, Hanack K. Development of an optimized cell-based selection system for phage display libraries. Biol Methods Protoc 2025; 10:bpaf009. [PMID: 39968222 PMCID: PMC11835232 DOI: 10.1093/biomethods/bpaf009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2024] [Revised: 01/08/2025] [Accepted: 02/05/2025] [Indexed: 02/20/2025] Open
Abstract
The discovery of antibodies through phage display is significantly influenced by antigen presentation during panning, particularly for membrane-anchored proteins, which pose challenges due to their complex structures. Traditional approaches, such as whole cells expressing the target protein, often result in low antigen density and high background signals. In this study, we describe an alternative method using stably transfected cell lines that express the target antigen on their surface, regulated by an intracellular enhanced green fluorescent protein (EGFP) signal. This system enables high-throughput flow cytometry-based screening of phage display libraries to isolate human antibodies that recognize the native conformation of membrane proteins. Using human epithelial cell adhesion molecule (EpCAM) and human neuroplastin 65 (NP65) as model antigens, we established an optimized screening workflow with polyclonal phage pools. Selected EpCAM-specific single-chain variable fragments (scFvs) from a naïve library were recombinantly expressed with an IgG4 scaffold and characterized for specific binding. This approach provides an effective platform for the identification of antibodies against membrane proteins in their native state.
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Affiliation(s)
- Malgorzata Czarnecka
- Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam, Germany
| | - Nicole Findik
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
| | - Anja Schlör
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
| | - Katja Hanack
- Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam, Germany
- new/era/mabs GmbH, August-Bebel-Str. 89, 14482 Potsdam, Germany
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6
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Gorelik M, Miersch S, Sidhu SS. Structural Survey of Antigen Recognition by Synthetic Human Antibodies. Cold Spring Harb Protoc 2025; 2025:pdb.over107759. [PMID: 38594044 DOI: 10.1101/pdb.over107759] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/11/2024]
Abstract
Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.
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Affiliation(s)
- Maryna Gorelik
- School of Pharmacy, University of Waterloo, Waterloo, Ontario N2G 1C5, Canada
| | - Shane Miersch
- School of Pharmacy, University of Waterloo, Waterloo, Ontario N2G 1C5, Canada
| | - Sachdev S Sidhu
- School of Pharmacy, University of Waterloo, Waterloo, Ontario N2G 1C5, Canada
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7
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Lim CC, Lim TS. Profiling the broad antibody diversity of lymphatic filariasis immune antibody repertoire by deep sequencing. Int J Biol Macromol 2025; 290:140037. [PMID: 39828167 DOI: 10.1016/j.ijbiomac.2025.140037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2024] [Revised: 01/16/2025] [Accepted: 01/16/2025] [Indexed: 01/22/2025]
Abstract
Lymphatic filariasis is caused by infections of thread-like filarial worms, namely Wuchereria bancrofti, Brugia Malayi and Brugia timori. However, in-depth analysis of the antibody repertoire against Lymphatic filariasis is lacking. Using high-throughput sequencing of antibody repertoires, immunome analysis of IgG (LG) and IgM (LM) repertoires were studied. Despite significant differences between LG and LM in V(D)J gene usage, IGHV4-34, IGHV6-1, IGHD3-10 and IGHJ4 were preferred in both repertoires. The CDR3 in the LG repertoire showed a longer length than LM. Higher SHM level were observed in LG sequences and presence of oligoclonal sequences indicates the extent of clonal expansion. The prevalence of rare clonotypes in LM repertoire depicts the high clonal diversity when compared to LG repertoire. Monoclonal antibodies against closely related parasitic infections were present within the LG repertoire suggesting that immune repertoires may not be as exclusive and biased against the target infection as initially thought. The characterization of the immune repertoire can provide critical insight into the antibody response patterns in disease state, antibody generation process during infections and future antibody designs.
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Affiliation(s)
- Chia Chiu Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia
| | - Theam Soon Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia; Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Penang, Malaysia.
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8
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Yadav AJ, Bhagat K, Sharma A, Padhi AK. Navigating the landscape: A comprehensive overview of computational approaches in therapeutic antibody design and analysis. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2025; 144:33-76. [PMID: 39978970 DOI: 10.1016/bs.apcsb.2024.10.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/22/2025]
Abstract
Immunotherapy, harnessing components like antibodies, cells, and cytokines, has become a cornerstone in treating diseases such as cancer and autoimmune disorders. Therapeutic antibodies, in particular, have transformed modern medicine, providing a targeted approach that destroys disease-causing cells while sparing healthy tissues, thereby reducing the side effects commonly associated with chemotherapy. Beyond oncology, these antibodies also hold promise in addressing chronic infections where conventional therapeutics may fall short. However, antibodies identified through in vivo or in vitro methods often require extensive engineering to enhance their therapeutic potential. This optimization process, aimed at improving affinity, specificity, and reducing immunogenicity, is both challenging and costly, often involving trade-offs between critical properties. Traditional methods of antibody development, such as hybridoma technology and display techniques, are resource-intensive and time-consuming. In contrast, computational approaches offer a faster, more efficient alternative, enabling the precise design and analysis of therapeutic antibodies. These methods include sequence and structural bioinformatics approaches, next-generation sequencing-based data mining, machine learning algorithms, systems biology, immuno-informatics, and integrative approaches. These approaches are advancing the field by providing new insights and enhancing the accuracy of antibody design and analysis. In conclusion, computational approaches are essential in the development of therapeutic antibodies, significantly improving the precision and speed of discovery, optimization, and validation. Integrating these methods with experimental approaches accelerates therapeutic antibody development, paving the way for innovative strategies and treatments for various diseases ranging from cancers to autoimmune and infectious diseases.
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Affiliation(s)
- Amar Jeet Yadav
- Laboratory for Computational Biology & Biomolecular Design, School of Biochemical Engineering, Indian Institute of Technology (BHU) Varanasi, Varanasi, Uttar Pradesh, India
| | - Khushboo Bhagat
- Laboratory for Computational Biology & Biomolecular Design, School of Biochemical Engineering, Indian Institute of Technology (BHU) Varanasi, Varanasi, Uttar Pradesh, India
| | - Akshit Sharma
- Laboratory for Computational Biology & Biomolecular Design, School of Biochemical Engineering, Indian Institute of Technology (BHU) Varanasi, Varanasi, Uttar Pradesh, India
| | - Aditya K Padhi
- Laboratory for Computational Biology & Biomolecular Design, School of Biochemical Engineering, Indian Institute of Technology (BHU) Varanasi, Varanasi, Uttar Pradesh, India.
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9
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Jacobs BM, Gasperi C, Kalluri SR, Al-Najjar R, McKeon MO, Else J, Pukaj A, Held F, Sawcer S, Ban M, Hemmer B. Single-cell analysis of cerebrospinal fluid reveals common features of neuroinflammation. Cell Rep Med 2025; 6:101733. [PMID: 39708811 PMCID: PMC11866449 DOI: 10.1016/j.xcrm.2024.101733] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 04/26/2024] [Accepted: 08/19/2024] [Indexed: 12/23/2024]
Abstract
Neuroinflammation is often characterized by immune cell infiltrates in the cerebrospinal fluid (CSF). Here, we apply single-cell RNA sequencing to explore the functional characteristics of these cells in patients with various inflammatory, infectious, and non-inflammatory neurological disorders. We show that CSF is distinct from the peripheral blood in terms of both cellular composition and gene expression. We report that the cellular and transcriptional landscape of CSF is altered in neuroinflammation but is strikingly similar across different neuroinflammatory disorders. We find clonal expansion of CSF lymphocytes in all disorders but most pronounced in inflammatory diseases, and we functionally characterize the transcriptional features of these cells. Finally, we explore the genetic control of gene expression in CSF lymphocytes. Our results highlight the common features of immune cells in the CSF compartment across diverse neurological diseases and may help to identify new targets for drug development or repurposing in multiple sclerosis (MS).
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Affiliation(s)
- Benjamin M Jacobs
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK; Wolfson Institute of Population Health, Queen Mary University of London, London, UK
| | - Christiane Gasperi
- Department of Neurology, Technical University of Munich, Munich, Germany
| | | | - Raghda Al-Najjar
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
| | - Mollie O McKeon
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
| | - Jonathan Else
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
| | - Albert Pukaj
- Department of Neurology, Technical University of Munich, Munich, Germany
| | - Friederike Held
- Department of Neurology, Technical University of Munich, Munich, Germany
| | - Stephen Sawcer
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.
| | - Maria Ban
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.
| | - Bernhard Hemmer
- Department of Neurology, Technical University of Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
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10
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Camus V, Viennot M, Viailly PJ, Drieux F, Veresezan EL, Bobée V, Rainville V, Bohers E, Sesques P, Haioun C, Durot E, Bayaram M, Rossi C, Martin L, Penther D, Kaltenbach S, Bruneau J, Paillassa J, Tournilhac O, Gower N, Willaume A, Antier C, Renaud L, Lévêque E, Decazes P, Becker S, Tonnelet D, Gaulard P, Tilly H, Molina TJ, Traverse-Glehen A, Donzel M, Ruminy P, Jardin F. Identification of primary mediastinal B-cell lymphomas with higher clonal dominance and poorer outcome using 5'RACE. Blood Adv 2025; 9:101-115. [PMID: 39293080 PMCID: PMC11742575 DOI: 10.1182/bloodadvances.2024013723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2024] [Revised: 08/30/2024] [Accepted: 09/03/2024] [Indexed: 09/20/2024] Open
Abstract
ABSTRACT There is a scarcity of data on the tumor B-cell receptor (BCR) repertoire and lymphoid microenvironment in primary mediastinal B-cell lymphoma (PMBL). We applied 5' rapid amplification of complimentary DNA ends (5'RACE) to tumor RNA samples from 137 patients with PMBL with available gene expression profiling and next-generation sequencing data. We obtained 5'RACE results for 75 of the 137 (54.7%) patients with the following clinical characteristics: median age (range), 33 years (18-64); female, 53.3%; performance status score 0 to 1, 86.7%; stage I to II, 57.3%; first-line treatment with anti-CD20 plus doxorubicin-based chemotherapy, 100%. Among the 60 biopsies that expressed a productive BCR, we highlighted a strong somatic hypermutation profile, defined as <98% identity to the germ line sequence, with 58 (96.7%) patients carrying mutated IgVH. We then identified a subgroup of 12 of the 75 patients (16%) with a worse prognosis (progression-free survival [PFS]: hazard ratio [HR], 17; overall survival [OS]: HR, 21) that was associated with the highest clonal dominance (HCD) status, defined as the dominant clonotype representing >81.1% and >78.6% of all complementarity-determining region 3 sequences for IgVH and IgVL, respectively. When compared with other patients, this subgroup had similar clinical characteristics but a greater median allele frequency for all somatic variants, a decreased BCR diversity, and greater expression of PDL1/PDL2 and MS4A1 genes, suggesting greater tumoral infiltration. We confirmed this poorer prognosis in a multivariate model and in an independent validation cohort in which 6 of 37 (16%) PMBL patients exhibited HCD (PFS: HR, 12; OS: HR, 17).
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Affiliation(s)
- Vincent Camus
- Department of Hematology, Centre Henri Becquerel, Rouen, France
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Mathieu Viennot
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | | | - Fanny Drieux
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
- Department of Pathology, Centre Henri Becquerel, Rouen, France
| | - Elena-Liana Veresezan
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
- Department of Pathology, Centre Henri Becquerel, Rouen, France
| | - Victor Bobée
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
- Biological Hematology Laboratory, Centre Hospitalier Universitaire de Rouen, Hôpital Charles Nicolle, Rouen, France
| | - Vinciane Rainville
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Elodie Bohers
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Pierre Sesques
- Department of Hematology, Hospices Civils de Lyon, Pierre-Bénite, France
| | - Corinne Haioun
- Department of Lymphoid Malignancies, Centre Hospitalier Universitaire Henri Mondor, Assistance Publique-Hôpitaux de Paris, Créteil, France
| | - Eric Durot
- Department of Hematology, Centre Hospitalier Universitaire de Reims, Reims, France
| | - Michael Bayaram
- Department of Pathology, Centre Hospitalier Universitaire de Reims, Reims, France
| | - Cédric Rossi
- Department of Hematology, Centre Hospitalier Universitaire de Dijon, Dijon, France
| | - Laurent Martin
- Department of Pathology, Centre Hospitalier Universitaire de Dijon, Dijon, France
| | - Dominique Penther
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
- Laboratory of Genetic Oncology, Centre Henri Becquerel, Rouen, France
| | - Sophie Kaltenbach
- Centre Hospitalier Universitaire Necker, Laboratory of Onco-Hematology, Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Julie Bruneau
- University of Paris, Institut Imagine, Institut des Maladies Génétiques, INSERM UMR1163, Paris, France
- Department of Pathology, Université Paris Cité, Assistance Publique-Hôpitaux de Paris, Hôpitaux Necker et Robert Debré, Paris, France
| | - Jérôme Paillassa
- Department of Hematology, Centre Hospitalier Universitaire d’Angers, Angers, France
| | - Olivier Tournilhac
- Department of Hematology, Centre Hospitalier Universitaire de Clermont-Ferrand, Clermont-Ferrand, France
| | - Nicolas Gower
- Department of Hematology, Centre Hospitalier Universitaire de Lille, Hôpital Claude Hurriez, Lille, France
| | - Alexandre Willaume
- Department of Hematology, Hôpital Saint Vincent de Paul, Groupe Hospitalier de l'Institut Catholique de Lille, Lille, France
| | - Chloé Antier
- Department of Hematology, Centre Hospitalier Universitaire de Nantes, Nantes, France
| | - Loïc Renaud
- Department of Hematology, Gustave Roussy, Villejuif, France
| | - Emilie Lévêque
- Clinical Research Unit, Centre Henri Becquerel, Rouen, France
| | - Pierre Decazes
- Department of Nuclear Medicine and QuantIF-LITIS-EA4108, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Stéphanie Becker
- Department of Pathology, Centre Hospitalier Universitaire Henri Mondor, Assistance Publique-Hôpitaux de Paris, Créteil, France
| | - David Tonnelet
- Department of Nuclear Medicine and QuantIF-LITIS-EA4108, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Philippe Gaulard
- Department of Pathology, Centre Hospitalier Universitaire Henri Mondor, Assistance Publique-Hôpitaux de Paris, Créteil, France
| | - Hervé Tilly
- Department of Hematology, Centre Henri Becquerel, Rouen, France
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Thierry Jo Molina
- University of Paris, Institut Imagine, Institut des Maladies Génétiques, INSERM UMR1163, Paris, France
- Department of Pathology, Université Paris Cité, Assistance Publique-Hôpitaux de Paris, Hôpitaux Necker et Robert Debré, Paris, France
| | | | - Marie Donzel
- Department of Pathology, Hospices Civils de Lyon, Université Lyon 1, Pierre-Bénite, France
| | - Philippe Ruminy
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
| | - Fabrice Jardin
- Department of Hematology, Centre Henri Becquerel, Rouen, France
- INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France
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11
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Istomina PV, Gorchakov AA, Paoin C, Yamabhai M. Phage display for discovery of anticancer antibodies. N Biotechnol 2024; 83:205-218. [PMID: 39186973 DOI: 10.1016/j.nbt.2024.08.506] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2024] [Revised: 08/20/2024] [Accepted: 08/20/2024] [Indexed: 08/28/2024]
Abstract
Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development.
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Affiliation(s)
- Polina V Istomina
- Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand
| | - Andrey A Gorchakov
- Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences, Lavrentieva 8/2, Novosibirsk 630090, Russia
| | - Chatchanok Paoin
- Medical Oncology Division, Institute of Medicine, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand
| | - Montarop Yamabhai
- Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Suranaree, Muang, 111 University Avenue, Nakhon Ratchasima 30000, Thailand.
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12
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Naylon SH, Richaud AD, Zhao G, Bui L, Dufresne CP, Wu CJ, Wangpaichitr M, Savaraj N, Roche SP. A platform of ADAPTive scaffolds: development of CDR-H3 β-hairpin mimics into covalent inhibitors of the PD1/PDL1 immune checkpoint. RSC Chem Biol 2024; 5:d4cb00174e. [PMID: 39552936 PMCID: PMC11562385 DOI: 10.1039/d4cb00174e] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2024] [Accepted: 10/30/2024] [Indexed: 11/19/2024] Open
Abstract
Aberrant and dysregulated protein-protein interactions (PPIs) drive a significant number of human diseases, which is why they represent a major class of targets in drug discovery. Although a number of high-affinity antibody-based drugs have emerged in this therapeutic space, the discovery of smaller PPI inhibitors is lagging far behind, underscoring the need for novel scaffold modalities. To bridge this gap, we introduce a biomimetic platform technology - adaptive design of antibody paratopes into therapeutics (ADAPT) - that enables the paratope-forming binding loops of antibodies to be crafted into large β-hairpin scaffolds (ADAPTins). In this study, we describe a novel strategy for engineering native CDR-H3 "hot loops" with varying sequences, lengths, and rigidity into ADAPTins, ultimately transforming these compounds into irreversible covalent inhibitors. A proof-of-concept was established by creating a series of ADAPTin blockers of the PD1:PDL1 immune checkpoint PPI (blocking activity EC50 < 0.3 μM) which were subsequently modified into potent covalent PD1 inhibitors. The compelling rate of stable and folded ADAPTins above physiological temperature (21 out of 29) obtained across six different scaffolds suggests that the platform technology could provide a novel opportunity for high-quality peptide display and biological screening.
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Affiliation(s)
- Sarah H Naylon
- Department of Chemistry and Biochemistry, Florida Atlantic University Boca Raton Florida 33431 USA
| | - Alexis D Richaud
- Department of Chemistry and Biochemistry, Florida Atlantic University Boca Raton Florida 33431 USA
| | - Guangkuan Zhao
- Department of Chemistry and Biochemistry, Florida Atlantic University Boca Raton Florida 33431 USA
| | - Linda Bui
- Department of Chemistry and Biochemistry, Florida Atlantic University Boca Raton Florida 33431 USA
| | | | - Chunjing J Wu
- University of Miami, Miller School of Medicine Miami Florida 33136 USA
| | | | - Niramol Savaraj
- University of Miami, Miller School of Medicine Miami Florida 33136 USA
| | - Stéphane P Roche
- Department of Chemistry and Biochemistry, Florida Atlantic University Boca Raton Florida 33431 USA
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13
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Zhao G, Richaud AD, Williamson RT, Feig M, Roche SP. De Novo Synthesis and Structural Elucidation of CDR-H3 Loop Mimics. ACS Chem Biol 2024; 19:1583-1592. [PMID: 38916527 PMCID: PMC11299430 DOI: 10.1021/acschembio.4c00236] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/26/2024]
Abstract
The binding affinity of antibodies to specific antigens stems from a remarkably broad repertoire of hypervariable loops known as complementarity-determining regions (CDRs). While recognizing the pivotal role of the heavy-chain 3 CDRs (CDR-H3s) in maximizing antibody-antigen affinity and specificity, the key structural determinants responsible for their adaptability to diverse loop sequences, lengths, and noncanonical structures are hitherto unknown. To address this question, we achieved a de novo synthesis of bulged CDR-H3 mimics excised from their full antibody context. CD and NMR data revealed that these stable standalone β-hairpin scaffolds are well-folded and retain many of the native bulge CDR-H3 features in water. In particular, the tryptophan residue, highly conserved across CDR-H3 sequences, was found to extend the kinked base of these β-bulges through a combination of stabilizing intramolecular hydrogen bond and CH/π interaction. The structural ensemble consistent with our NMR observations exposed the dynamic nature of residues at the base of the loop, suggesting that β-bulges act as molecular hinges connecting the rigid stem to the more flexible loops of CDR-H3s. We anticipate that this deeper structural understanding of CDR-H3s will lay the foundation to inform the design of antibody drugs broadly and engineer novel CDR-H3 peptide scaffolds as therapeutics.
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Affiliation(s)
- Guangkuan Zhao
- Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United States
| | - Alexis D. Richaud
- Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United States
| | - R. Thomas Williamson
- Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, NC 28409, United States
| | - Michael Feig
- Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, United States
| | - Stéphane P. Roche
- Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United States
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14
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Strazza V, Rossi M, Avati A, Tiseo G, Falcone M, Cusi MG, Menichetti F, Ricciardi-Castagnoli P, Tinti C, Pileri P. Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology. Front Immunol 2024; 15:1341389. [PMID: 38698845 PMCID: PMC11064063 DOI: 10.3389/fimmu.2024.1341389] [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: 11/20/2023] [Accepted: 03/06/2024] [Indexed: 05/05/2024] Open
Abstract
Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.
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Affiliation(s)
- Veronica Strazza
- Hyper Antibody Research & Development (HARD) -Lab, Toscana Life Sciences Foundation, Siena, Italy
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
| | - Marco Rossi
- Hyper Antibody Research & Development (HARD) -Lab, Toscana Life Sciences Foundation, Siena, Italy
| | - Andrea Avati
- Hyper Antibody Research & Development (HARD) -Lab, Toscana Life Sciences Foundation, Siena, Italy
| | - Giusy Tiseo
- Infectious Diseases Unit, Department of Clinical and Experimental Medicine, Azienda Ospedaliero Universitaria Pisana, University of Pisa, Pisa, Italy
| | - Marco Falcone
- Infectious Diseases Unit, Department of Clinical and Experimental Medicine, Azienda Ospedaliero Universitaria Pisana, University of Pisa, Pisa, Italy
| | - Maria Grazia Cusi
- Department of Medical Biotechnologies, University of Siena, Siena, Italy
| | - Francesco Menichetti
- Infectious Diseases Unit, Department of Clinical and Experimental Medicine, Azienda Ospedaliero Universitaria Pisana, University of Pisa, Pisa, Italy
| | | | - Cristina Tinti
- Hyper Antibody Research & Development (HARD) -Lab, Toscana Life Sciences Foundation, Siena, Italy
| | - Piero Pileri
- Hyper Antibody Research & Development (HARD) -Lab, Toscana Life Sciences Foundation, Siena, Italy
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15
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Beaulaurier J, Ly L, Duty JA, Tyer C, Stevens C, Hung CT, Sookdeo A, Drong AW, Kowdle S, Turner DJ, Juul S, Hickey S, Lee B. De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.26.586834. [PMID: 38585716 PMCID: PMC10996687 DOI: 10.1101/2024.03.26.586834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2024]
Abstract
Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.
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16
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Deng Y, Tang M, Ross TM, Schmidt AG, Chakraborty AK, Lingwood D. Repeated vaccination with homologous influenza hemagglutinin broadens human antibody responses to unmatched flu viruses. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2024:2024.03.27.24303943. [PMID: 38585939 PMCID: PMC10996724 DOI: 10.1101/2024.03.27.24303943] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2024]
Abstract
The on-going diversification of influenza virus necessicates annual vaccine updating. The vaccine antigen, the viral spike protein hemagglutinin (HA), tends to elicit strain-specific neutralizing activity, predicting that sequential immunization with the same HA strain will boost antibodies with narrow coverage. However, repeated vaccination with homologous SARS-CoV-2 vaccine eventually elicits neutralizing activity against highly unmatched variants, questioning this immunological premise. We evaluated a longitudinal influenza vaccine cohort, where each year the subjects received the same, novel H1N1 2009 pandemic vaccine strain. Repeated vaccination gradually enhanced receptor-blocking antibodies (HAI) to highly unmatched H1N1 strains within individuals with no initial memory recall against these historical viruses. An in silico model of affinity maturation in germinal centers integrated with a model of differentiation and expansion of memory cells provides insight into the mechanisms underlying these results and shows how repeated exposure to the same immunogen can broaden the antibody response against diversified targets.
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17
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Shingai M, Iida S, Kawai N, Kawahara M, Sekiya T, Ohno M, Nomura N, Handabile C, Kawakita T, Omori R, Yamagishi J, Sano K, Ainai A, Suzuki T, Ohnishi K, Ito K, Kida H. Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire. J Virol 2024; 98:e0199523. [PMID: 38323813 PMCID: PMC10949447 DOI: 10.1128/jvi.01995-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Accepted: 01/14/2024] [Indexed: 02/08/2024] Open
Abstract
Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.
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Affiliation(s)
- Masashi Shingai
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
- Division of Vaccine Immunology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Sayaka Iida
- Division of Bioinformatics, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Naoko Kawai
- Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Mamiko Kawahara
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Toshiki Sekiya
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
- International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Marumi Ohno
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
- One Health Research Center, Hokkaido University, Sapporo, Japan
| | - Naoki Nomura
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
| | - Chimuka Handabile
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
| | - Tomomi Kawakita
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
- Division of Vaccine Immunology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Ryosuke Omori
- Division of Bioinformatics, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Junya Yamagishi
- International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Kaori Sano
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Akira Ainai
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tadaki Suzuki
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuo Ohnishi
- Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kimihito Ito
- International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Division of Bioinformatics, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Hiroshi Kida
- Division of Biologics Development, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
- International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
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18
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Gallo E. The rise of big data: deep sequencing-driven computational methods are transforming the landscape of synthetic antibody design. J Biomed Sci 2024; 31:29. [PMID: 38491519 PMCID: PMC10943851 DOI: 10.1186/s12929-024-01018-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 03/05/2024] [Indexed: 03/18/2024] Open
Abstract
Synthetic antibodies (Abs) represent a category of artificial proteins capable of closely emulating the functions of natural Abs. Their in vitro production eliminates the need for an immunological response, streamlining the process of Ab discovery, engineering, and development. These artificially engineered Abs offer novel approaches to antigen recognition, paratope site manipulation, and biochemical/biophysical enhancements. As a result, synthetic Abs are fundamentally reshaping conventional methods of Ab production. This mirrors the revolution observed in molecular biology and genomics as a result of deep sequencing, which allows for the swift and cost-effective sequencing of DNA and RNA molecules at scale. Within this framework, deep sequencing has enabled the exploration of whole genomes and transcriptomes, including particular gene segments of interest. Notably, the fusion of synthetic Ab discovery with advanced deep sequencing technologies is redefining the current approaches to Ab design and development. Such combination offers opportunity to exhaustively explore Ab repertoires, fast-tracking the Ab discovery process, and enhancing synthetic Ab engineering. Moreover, advanced computational algorithms have the capacity to effectively mine big data, helping to identify Ab sequence patterns/features hidden within deep sequencing Ab datasets. In this context, these methods can be utilized to predict novel sequence features thereby enabling the successful generation of de novo Ab molecules. Hence, the merging of synthetic Ab design, deep sequencing technologies, and advanced computational models heralds a new chapter in Ab discovery, broadening our comprehension of immunology and streamlining the advancement of biological therapeutics.
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Affiliation(s)
- Eugenio Gallo
- Department of Medicinal Chemistry, Avance Biologicals, 950 Dupont Street, Toronto, ON, M6H 1Z2, Canada.
- Department of Protein Engineering, RevivAb, Av. Ipiranga, 6681, Partenon, Porto Alegre, RS, 90619-900, Brazil.
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19
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Porebski BT, Balmforth M, Browne G, Riley A, Jamali K, Fürst MJLJ, Velic M, Buchanan A, Minter R, Vaughan T, Holliger P. Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening. Nat Biomed Eng 2024; 8:214-232. [PMID: 37814006 PMCID: PMC10963267 DOI: 10.1038/s41551-023-01093-3] [Citation(s) in RCA: 24] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2022] [Accepted: 08/23/2023] [Indexed: 10/11/2023]
Abstract
Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody-antigen interactions. The method, which we named 'deep screening', involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument's flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.
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Affiliation(s)
| | | | | | - Aidan Riley
- Biologics Engineering, AstraZeneca, Cambridge, UK
| | | | - Maximillian J L J Fürst
- MRC Laboratory of Molecular Biology, Cambridge, UK
- Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands
| | | | | | - Ralph Minter
- Biologics Engineering, AstraZeneca, Cambridge, UK
- Alchemab Therapeutics, London, UK
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20
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De Souza Cordeiro LM, Atkinson KC, Aivazian A, Joyce PF, Jia F, Mascioni A. Electrostatic properties of human germlines and biodistribution of small biologics. MAbs 2024; 16:2311991. [PMID: 38334129 PMCID: PMC10860348 DOI: 10.1080/19420862.2024.2311991] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Accepted: 01/25/2024] [Indexed: 02/10/2024] Open
Abstract
Off-target biodistribution of biologics bears important toxicological consequences. Antibody fragments intended for use as vectors of cytotoxic payloads (e.g. antibody-drug conjugates, radiotherapy) can accumulate at clearance organs like kidneys and liver, where they can cause dose-limiting toxicities. Renal and hepatic uptakes are known to be affected by protein electrostatics, which promote protein internalization through pinocytosis. Using minibodies as a model of an antibody fragment lacking FcRn recycling, we compared the biodistributions of leads with different degrees of accumulation at the kidney and liver. We identified a positive electrostatic patch highly conserved in a germline family very commonly used in the humanization of approved biologics. Neutralization of this patch led to a drastic reduction in the kidney uptake, leading to a biodistribution more favorable to the delivery of highly cytotoxic payloads. Next, we conducted a high throughput study of the electrostatic properties for all combinations of VH and VL germlines. This analysis shows how different VH/VL combinations exhibit varying tendencies to create electrostatic patches, resulting in Fv variants with different isoelectric points. Our work emphasizes the importance of carefully selecting germlines for humanization with optimal electrostatic properties in order to control the unspecific tissue uptake of low molecular weight biologics.
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Affiliation(s)
| | | | - Argin Aivazian
- Preclinical discovery, ImaginAb, Inc, Inglewood, CA, USA
| | | | - Fang Jia
- Preclinical discovery, ImaginAb, Inc, Inglewood, CA, USA
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21
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Lobos CA, Chatzileontiadou DSM, Sok B, Almedia C, Halim H, D'Orsogna L, Gras S. Molecular insights into the HLA-B35 molecules' classification associated with HIV control. Immunol Cell Biol 2024; 102:34-45. [PMID: 37811811 PMCID: PMC10952751 DOI: 10.1111/imcb.12698] [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: 10/14/2022] [Revised: 07/04/2023] [Accepted: 09/19/2023] [Indexed: 10/10/2023]
Abstract
Human leukocyte antigen (HLA) class I molecules have been shown to influence the immune response to HIV infection and acquired immunodeficiency syndrome progression. Polymorphisms within the HLA-B35 molecules divide the family into two groups, namely, Px and PY. The Px group is associated with deleterious effects and accelerated disease progression in HIV+ patients, whereas the PY group is not. The classification is based on the preferential binding of a tyrosine at the C-terminal part of the peptide in the PY group, and a nontyrosine residue in the Px group. However, there is a lack of knowledge on the molecular differences between the two groups. Here, we have investigated three HLA-B35 molecules, namely, HLA-B*35:01 (PY), HLA-B*35:03 (Px) and HLA-B*35:05 (unclassified). We selected an HIV-derived peptide, NY9, and demonstrated that it can trigger a polyfunctional CD8+ T-cell response in HLA-B*35:01+ /HIV+ patients. We determined that in the complex with the NY9 peptide, the PY molecule was more stable than the Px molecule. We solved the crystal structures of the three HLA molecules in complex with the NY9 peptide, and structural similarities with HLA-B*35:01 would classify the HLA-B*35:05 within the PY group. Interestingly, we found that HLA-B*35:05 can also bind a small molecule in its cleft, suggesting that small drugs could bind as well.
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Affiliation(s)
- Christian A Lobos
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular ScienceLa Trobe UniversityBundooraVICAustralia
- Department of Biochemistry and Molecular BiologyMonash UniversityClaytonVICAustralia
| | - Demetra SM Chatzileontiadou
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular ScienceLa Trobe UniversityBundooraVICAustralia
- Department of Biochemistry and Molecular BiologyMonash UniversityClaytonVICAustralia
| | - Bonin Sok
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular ScienceLa Trobe UniversityBundooraVICAustralia
| | - Coral‐Ann Almedia
- Department of Clinical Immunology and PathWestFiona Stanley HospitalPerthWAAustralia
- School of MedicineUniversity of Western AustraliaPerthWAAustralia
| | - Hanim Halim
- Department of Biochemistry and Molecular BiologyMonash UniversityClaytonVICAustralia
| | - Lloyd D'Orsogna
- Department of Clinical Immunology and PathWestFiona Stanley HospitalPerthWAAustralia
- School of MedicineUniversity of Western AustraliaPerthWAAustralia
| | - Stephanie Gras
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular ScienceLa Trobe UniversityBundooraVICAustralia
- Department of Biochemistry and Molecular BiologyMonash UniversityClaytonVICAustralia
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22
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Levi R, Dvorkin S, Louzoun Y. Shared bias in H chain V-J pairing in naive and memory B cells. Front Immunol 2023; 14:1166116. [PMID: 37790930 PMCID: PMC10543446 DOI: 10.3389/fimmu.2023.1166116] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Accepted: 08/23/2023] [Indexed: 10/05/2023] Open
Abstract
Introduction H chain rearrangement in B cells is a two-step process where first DH binds JH, and only then VH is joined to the complex. As such, there is no direct rearrangement between VH and JH. Results Nevertheless, we here show that the VHJH combinations frequency in humans deviates from the one expected based on each gene usage frequency. This bias is observed mainly in functional rearrangements, and much less in out-of-frame rearrangements. The bias cannot be explained by preferred binding for DH genes or a preferred reading frame. Preferred VH JH combinations are shared between donors. Discussion These results suggest a common structural mechanism for these biases. Through development, thepreferred VH JH combinations evolve during peripheral selection to become stronger, but less shared. We propose that peripheral Heavy chain VH JH usage is initially shaped by a structural selection before the naive B cellstate, followed by pathogen-induced selection for host specific VH-JH pairs.
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Affiliation(s)
| | | | - Yoram Louzoun
- Department of Mathematics, Bar Ilan University, Ramat Gan, Israel
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23
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Ronsard L, Yousif AS, Nait Mohamed FA, Feldman J, Okonkwo V, McCarthy C, Schnabel J, Caradonna T, Barnes RM, Rohrer D, Lonberg N, Schmidt A, Lingwood D. Engaging an HIV vaccine target through the acquisition of low B cell affinity. Nat Commun 2023; 14:5249. [PMID: 37640732 PMCID: PMC10462694 DOI: 10.1038/s41467-023-40918-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 08/16/2023] [Indexed: 08/31/2023] Open
Abstract
Low affinity is common for germline B cell receptors (BCR) seeding development of broadly neutralizing antibodies (bnAbs) that engage hypervariable viruses, including HIV. Antibody affinity selection is also non-homogenizing, insuring the survival of low affinity B cell clones. To explore whether this provides a natural window for expanding human B cell lineages against conserved vaccine targets, we deploy transgenic mice mimicking human antibody diversity and somatic hypermutation (SHM) and immunize with simple monomeric HIV glycoprotein envelope immunogens. We report an immunization regimen that focuses B cell memory upon the conserved CD4 binding site (CD4bs) through both conventional affinity maturation and reproducible expansion of low affinity BCR clones with public patterns in SHM. In the latter instance, SHM facilitates target acquisition by decreasing binding strength. This suggests that permissive B cell selection enables the discovery of antibody epitopes, in this case an HIV bnAb site.
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Affiliation(s)
- Larance Ronsard
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Ashraf S Yousif
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Faez Amokrane Nait Mohamed
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Jared Feldman
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Vintus Okonkwo
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Caitlin McCarthy
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Julia Schnabel
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Timothy Caradonna
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
| | - Ralston M Barnes
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA, 94063-2478, USA
| | - Daniel Rohrer
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA, 94063-2478, USA
| | - Nils Lonberg
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA, 94063-2478, USA
| | - Aaron Schmidt
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, 02115, USA
| | - Daniel Lingwood
- The Ragon Institute of Mass General, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA, 02139, USA.
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24
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Oksanen S, Saarinen R, Korkiakoski A, Lamminmäki U, Huovinen T. Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects. Sci Rep 2023; 13:13107. [PMID: 37567990 PMCID: PMC10421887 DOI: 10.1038/s41598-023-40241-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Accepted: 08/07/2023] [Indexed: 08/13/2023] Open
Abstract
Monoclonal antibodies (mAbs) and their fragments are widely used in therapeutics, diagnostics and basic research. Although display methods such as phage display offer high-throughput, affinities of individual antibodies need to be accurately measured in soluble format. We have developed a screening platform capable of providing genotyped functional data from a total of 9216 soluble, individual antigen binding fragment (Fab) clones by employing next-generation sequencing (NGS) with hierarchical indexing. Full-length, paired variable domain sequences (VL-VH) are linked to functional screening data, enabling in-depth analysis of mutation effects. The platform was applied to four phage display-selected scFv/Fab screening projects and one site-saturation VH affinity maturation project. Genotyped functional screening simultaneously enabled the identification of affinity improving mutations in the VH domain of Fab 49A3 recognizing Dengue virus non-structural protein 1 (NS1) serotype 2 and informed on VH residue positions which cannot be changed from wild-type without decreasing the affinity. Genotype-based identification revealed to us the extent of intraclonal signal variance inherent to single point screening data, a phenomenon often overlooked in the field. Moreover, genotyped screening eliminated the redundant selection of identical genotypes for further study and provided a new analysis tool to evaluate the success of phage display selections and remaining clonal diversity in the screened repertoires.
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Affiliation(s)
- Sami Oksanen
- Department of Life Sciences, University of Turku, 20520, Turku, Finland.
| | - Roope Saarinen
- Department of Life Sciences, University of Turku, 20520, Turku, Finland
| | | | - Urpo Lamminmäki
- Department of Life Sciences, University of Turku, 20520, Turku, Finland
| | - Tuomas Huovinen
- Department of Life Sciences, University of Turku, 20520, Turku, Finland.
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25
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Keppeke GD, Diogenes L, Gomes K, Andrade LEC. "Untargeting" autoantibodies using genome editing, a proof-of-concept study. Clin Immunol 2023; 251:109343. [PMID: 37094742 DOI: 10.1016/j.clim.2023.109343] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2023] [Revised: 04/11/2023] [Accepted: 04/13/2023] [Indexed: 04/26/2023]
Abstract
Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-α1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to ∼50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective antigens in T-gRNAs decreased ∼90% and ∼ 95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, suggesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases.
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Affiliation(s)
| | - Larissa Diogenes
- Rheumatology Division, Department of Medicine, Federal University of Sao Paulo, Brazil
| | - Kethellen Gomes
- Rheumatology Division, Department of Medicine, Federal University of Sao Paulo, Brazil
| | - Luis Eduardo Coelho Andrade
- Rheumatology Division, Department of Medicine, Federal University of Sao Paulo, Brazil; Immunology Division, Fleury Laboratory, Sao Paulo, Brazil.
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26
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Jiang R, Roy B, Wu Q, Mohanty S, Nowak RJ, Shaw AC, Kleinstein SH, O’Connor KC. The Plasma Cell Infiltrate Populating the Muscle Tissue of Patients with Inclusion Body Myositis Features Distinct B Cell Receptor Repertoire Properties. Immunohorizons 2023; 7:310-322. [PMID: 37171806 PMCID: PMC10579972 DOI: 10.4049/immunohorizons.2200078] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Accepted: 04/25/2023] [Indexed: 05/13/2023] Open
Abstract
Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.
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Affiliation(s)
- Roy Jiang
- Department of Immunobiology, Yale School of Medicine, New Haven, CT
| | - Bhaskar Roy
- Department of Neurology, Yale School of Medicine, New Haven, CT
| | - Qian Wu
- Department of Pathology, University of Connecticut School of Medicine, Farmington, CT
| | - Subhasis Mohanty
- Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT
| | | | - Albert C. Shaw
- Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT
| | - Steven H. Kleinstein
- Department of Immunobiology, Yale School of Medicine, New Haven, CT
- Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT
- Department of Pathology, Yale School of Medicine, New Haven, CT
| | - Kevin C. O’Connor
- Department of Immunobiology, Yale School of Medicine, New Haven, CT
- Department of Neurology, Yale School of Medicine, New Haven, CT
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27
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Xia M, Blazevic A, Fiore-Gartland A, Hoft DF. Impact of BCG vaccination on the repertoire of human γδ T cell receptors. Front Immunol 2023; 14:1100490. [PMID: 37056780 PMCID: PMC10089282 DOI: 10.3389/fimmu.2023.1100490] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 03/10/2023] [Indexed: 03/30/2023] Open
Abstract
Introduction Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a serious threat to human health. Vaccination with BCG prevents the development of the most severe forms of TB disease in infants and was recently shown to prevent Mtb infection in previously uninfected adolescents. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. However, our understanding of the effects of BCG vaccination on γδ T cell responses is incomplete. Methods In this study we performed γδ T cell receptor (TCR) repertoire sequencing of samples provided pre- and post-BCG vaccination from 10 individuals to identify specific receptors and TCR clones that are induced by BCG. Results Overall, there was no change in the diversity of γTCR or δTCR clonotypes in post- vs pre-BCG samples. Furthermore, the frequencies of TCR variable and joining region genes were minimally modulated by BCG vaccination at either the γTCR or δTCR loci. However, the γTCR and δTCR repertoires of individuals were highly dynamic; a median of ~1% of γTCR and ~6% of δTCR in the repertoire were found to significantly expand or contract in post- vs pre-BCG comparisons (FDR-q < 0.05). While many of the clonotypes whose frequency changed after BCG vaccination were not shared among multiple individuals in the cohort, several shared (i.e., "public") clonotypes were identified with a consistent increase or decrease in frequency across more than one individual; the degree of sharing of these clonotypes was significantly greater than the minimal sharing that would be expected among γTCR and δTCR repertoires. An in vitro analysis of Mtb antigen-reactive γδ T cells identified clonotypes that were similar or identical to the single-chain γTCRs and δTCRs that changed consistently after BCG vaccination; pairings of γTCRs and δTCRs that increased after BCG vaccination were significantly over-represented among the Mtb-reactive γδ T cells (p = 1.2e-6). Discussion These findings generate hypotheses about specific γδTCR clonotypes that may expand in response to BCG vaccination and may recognize Mtb antigens. Future studies are required to validate and characterize these clonotypes, with an aim to better understand the role of γδ T cells in Mtb immunity.
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Affiliation(s)
- Mei Xia
- Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, MO, United States
| | - Azra Blazevic
- Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, MO, United States
| | - Andrew Fiore-Gartland
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States
| | - Daniel F. Hoft
- Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, MO, United States
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, United States
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28
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Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS. Int J Mol Sci 2023; 24:ijms24065396. [PMID: 36982469 PMCID: PMC10049078 DOI: 10.3390/ijms24065396] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 01/27/2023] [Accepted: 03/08/2023] [Indexed: 03/14/2023] Open
Abstract
Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.TM-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries.
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29
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Potential of TCR sequencing in graft-versus-host disease. Bone Marrow Transplant 2023; 58:239-246. [PMID: 36477111 PMCID: PMC10005964 DOI: 10.1038/s41409-022-01885-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 11/18/2022] [Accepted: 11/22/2022] [Indexed: 12/12/2022]
Abstract
Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic haematopoietic stem cell transplantation (allo-HSCT). GvHD can occur in almost every tissue, with the skin, liver, and intestines being the mainly affected organs. T cells are implicated in initiating GvHD. T cells identify a broad range of antigens and mediate the immune response through receptors on their surfaces (T cell receptors, TCRs). The composition of TCRs within a T cell population defines the TCR repertoire of an individual, and this repertoire represents exposure to self and non-self proteins. Monitoring the changes in the TCR repertoire using TCR sequencing can provide an indication of the dynamics of a T cell population. Monitoring the frequency and specificities of specific TCR clonotypes longitudinally in different conditions and specimens (peripheral blood, GvHD-affected tissue samples) can provide insights into factors modulating immune reactions following allogeneic transplantation and will help to understand the underlying mechanisms mediating GvHD. This review provides insights into current studies of the TCR repertoire in GvHD and potential future clinical implications of TCR sequencing.
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30
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Hayashi S, Ishikawa S. Analyzing Antibody Repertoire Using Next-Generation Sequencing and Machine Learning. Methods Mol Biol 2023; 2552:465-473. [PMID: 36346609 DOI: 10.1007/978-1-0716-2609-2_26] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
Advances in high-throughput sequencing technologies have enabled comprehensive sequencing of the immune repertoire. Since repertoire analysis can help to explain the relationship between the immune system and diseases, several methods have been developed for repertoire analysis. Here, using simulated and real-world datasets, we describe how to use DeepRC, a method that applies cutting-edge machine learning techniques.
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Affiliation(s)
- Shuto Hayashi
- Department of Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Shumpei Ishikawa
- Department of Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
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31
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Steinke S, Roth KDR, Ruschig M, Langreder N, Polten S, Schneider KT, Ballmann R, Russo G, Zilkens KJK, Schubert M, Bertoglio F, Hust M. Antibody Selection via Phage Display in Microtiter Plates. Methods Mol Biol 2023; 2702:247-260. [PMID: 37679623 DOI: 10.1007/978-1-0716-3381-6_12] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/09/2023]
Abstract
The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection ("panning"), screening, and identification of monoclonal antibodies in less than 2 weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates.
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Affiliation(s)
- Stephan Steinke
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Kristian Daniel Ralph Roth
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Maximilian Ruschig
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Nora Langreder
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Saskia Polten
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Kai-Thomas Schneider
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Rico Ballmann
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Giulio Russo
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | | | - Maren Schubert
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
| | - Federico Bertoglio
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany
- Choose Life Biotech SA, Bellinzona, Switzerland
| | - Michael Hust
- Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany.
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32
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Zhong X, D’Antona AM. A potential antibody repertoire diversification mechanism through tyrosine sulfation for biotherapeutics engineering and production. Front Immunol 2022; 13:1072702. [PMID: 36569848 PMCID: PMC9774471 DOI: 10.3389/fimmu.2022.1072702] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Accepted: 11/23/2022] [Indexed: 12/13/2022] Open
Abstract
The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody's binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed.
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Luo S, Zhang J, Kreutzberger AJ, Eaton A, Edwards RJ, Jing C, Dai HQ, Sempowski GD, Cronin K, Parks R, Ye AY, Mansouri K, Barr M, Pishesha N, Williams AC, Vieira Francisco L, Saminathan A, Peng H, Batra H, Bellusci L, Khurana S, Alam SM, Montefiori DC, Saunders KO, Tian M, Ploegh H, Kirchhausen T, Chen B, Haynes BF, Alt FW. An antibody from single human V H-rearranging mouse neutralizes all SARS-CoV-2 variants through BA.5 by inhibiting membrane fusion. Sci Immunol 2022; 7:eadd5446. [PMID: 35951767 PMCID: PMC9407951 DOI: 10.1126/sciimmunol.add5446] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Accepted: 08/03/2022] [Indexed: 12/14/2022]
Abstract
SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact CDR3 sequences generated by nontemplated junctional modifications during V(D)J recombination. Immunizing this mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding motif via a CDR3-dominated recognition mode. Lattice light-sheet microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and nontraditional mechanism of action suggest that it might have therapeutic potential. Likewise, the SP1-77 binding epitope may inform vaccine strategies. Last, the type of humanized mouse models that we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.
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Affiliation(s)
- Sai Luo
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Jun Zhang
- Division of Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Alex J.B. Kreutzberger
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Amanda Eaton
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Robert J. Edwards
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Changbin Jing
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Hai-Qiang Dai
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Gregory D. Sempowski
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Kenneth Cronin
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Robert Parks
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Adam Yongxin Ye
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Katayoun Mansouri
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
| | - Maggie Barr
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - Novalia Pishesha
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Aimee Chapdelaine Williams
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Lucas Vieira Francisco
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Anand Saminathan
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Hanqin Peng
- Division of Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Himanshu Batra
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Lorenza Bellusci
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Surender Khurana
- Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - S. Munir Alam
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
| | - David C. Montefiori
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Surgery, Duke University, Durham, NC 27710, USA
| | - Kevin O. Saunders
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Surgery, Duke University, Durham, NC 27710, USA
- Department of Immunology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Ming Tian
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Hidde Ploegh
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Tom Kirchhausen
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Bing Chen
- Division of Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
| | - Barton F. Haynes
- Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Immunology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Frederick W. Alt
- Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
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The major role of junctional diversity in the horse antibody repertoire. Mol Immunol 2022; 151:231-241. [PMID: 36179605 DOI: 10.1016/j.molimm.2022.09.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2022] [Revised: 09/19/2022] [Accepted: 09/21/2022] [Indexed: 11/20/2022]
Abstract
The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.
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Sangesland M, Torrents de la Peña A, Boyoglu-Barnum S, Ronsard L, Mohamed FAN, Moreno TB, Barnes RM, Rohrer D, Lonberg N, Ghebremichael M, Kanekiyo M, Ward A, Lingwood D. Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus. Immunity 2022; 55:1693-1709.e8. [PMID: 35952670 PMCID: PMC9474600 DOI: 10.1016/j.immuni.2022.07.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Revised: 02/08/2022] [Accepted: 07/13/2022] [Indexed: 01/18/2023]
Abstract
Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) within their CDRH2 loops. Despite this, we demonstrated that both alleles encode for human IAV bnAbs that employ structurally convergent modes of contact to the same epitope. To resolve differences in lineage expandability, we compared F54 versus L54 as substrate within humanized mice, where antibodies develop with human-like CDRH3 diversity but are restricted to single VH genes. While both alleles encoded for bnAb precursors, only F54 IGHV1-69 supported elicitation of heterosubtypic serum bnAbs following immunization with a stalk-only nanoparticle vaccine. L54 IGHV1-69 was unproductive, co-encoding for anergic B cells and autoreactive stalk antibodies that were cleared from B cell memory. Moreover, human stalk antibodies also demonstrated L54-dependent autoreactivity. Therefore, IGHV1-69 polymorphism, which is skewed ethnically, gates tolerance and vaccine expandability of influenza bnAbs.
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Affiliation(s)
- Maya Sangesland
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA
| | - Alba Torrents de la Peña
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Seyhan Boyoglu-Barnum
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892-3005, USA
| | - Larance Ronsard
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA
| | - Faez Amokrane Nait Mohamed
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA
| | - Thalia Bracamonte Moreno
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA
| | - Ralston M Barnes
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA 94063-2478, USA
| | - Daniel Rohrer
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA 94063-2478, USA
| | - Nils Lonberg
- Bristol-Myers Squibb, 700 Bay Rd, Redwood City, CA 94063-2478, USA
| | - Musie Ghebremichael
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA
| | - Masaru Kanekiyo
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892-3005, USA
| | - Andrew Ward
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Daniel Lingwood
- The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA.
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Birley K, Leboreiro-Babe C, Rota EM, Buschhaus M, Gavriil A, Vitali A, Alonso-Ferrero M, Hopwood L, Parienti L, Ferry G, Flutter B, Himoudi N, Chester K, Anderson J. A novel anti-B7-H3 chimeric antigen receptor from a single-chain antibody library for immunotherapy of solid cancers. Mol Ther Oncolytics 2022; 26:429-443. [PMID: 36159778 PMCID: PMC9467911 DOI: 10.1016/j.omto.2022.08.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Accepted: 08/19/2022] [Indexed: 11/13/2022] Open
Abstract
B7-H3 (CD276) has emerged as a target for cancer immunotherapy by virtue of consistent expression in many malignancies, relative absence from healthy tissues, and an emerging role as a driver of tumor immune inhibition. Recent studies have reported B7-H3 to be a suitable target for chimeric antigen receptor-modified T cell (CAR-T) therapy using CARs constructed from established anti-B7-H3 antibodies converted into single-chain Fv format (scFv). We constructed and screened binders in an scFv library to generate a new anti-B7-H3 CAR-T with favorable properties. This allowed access to numerous specificities ready formatted for CAR evaluation. Selected anti-human B7-H3 scFvs were readily cloned into CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine, and proliferation assays. Two binders with divergent complementarity determining regions were found to show optimal antigen-specific cytotoxicity and cytokine secretion. One binder in second-generation CD28-CD3ζ CAR format induced sustained in vitro proliferation on repeat antigen challenge. The lead candidate CAR-T also demonstrated in vivo activity in a resistant neuroblastoma model. An empirical approach to B7-H3 CAR-T discovery through screening of novel scFv sequences in CAR-T format has led to the identification of a new construct with sustained proliferative capacity warranting further evaluation.
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Almagro JC, Mellado-Sánchez G, Pedraza-Escalona M, Pérez-Tapia SM. Evolution of Anti-SARS-CoV-2 Therapeutic Antibodies. Int J Mol Sci 2022; 23:ijms23179763. [PMID: 36077159 PMCID: PMC9456190 DOI: 10.3390/ijms23179763] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Revised: 08/20/2022] [Accepted: 08/25/2022] [Indexed: 01/17/2023] Open
Abstract
Since the first COVID-19 reports back in December of 2019, this viral infection caused by SARS-CoV-2 has claimed millions of lives. To control the COVID-19 pandemic, the Food and Drug Administration (FDA) and/or European Agency of Medicines (EMA) have granted Emergency Use Authorization (EUA) to nine therapeutic antibodies. Nonetheless, the natural evolution of SARS-CoV-2 has generated numerous variants of concern (VOCs) that have challenged the efficacy of the EUA antibodies. Here, we review the most relevant characteristics of these therapeutic antibodies, including timeline of approval, neutralization profile against the VOCs, selection methods of their variable regions, somatic mutations, HCDR3 and LCDR3 features, isotype, Fc modifications used in the therapeutic format, and epitope recognized on the receptor-binding domain (RBD) of SARS-CoV-2. One of the conclusions of the review is that the EUA therapeutic antibodies that still retain efficacy against new VOCs bind an epitope formed by conserved residues that seem to be evolutionarily conserved as thus, critical for the RBD:hACE-2 interaction. The information reviewed here should help to design new and more efficacious antibodies to prevent and/or treat COVID-19, as well as other infectious diseases.
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Affiliation(s)
- Juan C. Almagro
- GlobalBio, Inc., 320 Concord Ave, Cambridge, MA 02138, USA
- Unidad de Desarrollo e Investigación en Bioterapéuticos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos, LANSEIDI-FarBiotec-CONACyT, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Correspondence: (J.C.A.); (S.M.P.-T.)
| | - Gabriela Mellado-Sánchez
- Unidad de Desarrollo e Investigación en Bioterapéuticos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos, LANSEIDI-FarBiotec-CONACyT, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
| | - Martha Pedraza-Escalona
- CONACyT-Unidad de Desarrollo e Investigación en Bioterapéuticos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
| | - Sonia M. Pérez-Tapia
- Unidad de Desarrollo e Investigación en Bioterapéuticos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos, LANSEIDI-FarBiotec-CONACyT, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, Mexico City 11340, Mexico
- Correspondence: (J.C.A.); (S.M.P.-T.)
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Lee H, Yoo DK, Han J, Kim KH, Noh J, Lee Y, Lee E, Kwon S, Chung J. Optimization of peripheral blood volume for in silico reconstitution of the human B cell receptor repertoire. FEBS Open Bio 2022; 12:1634-1643. [PMID: 35866358 PMCID: PMC9433817 DOI: 10.1002/2211-5463.13467] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 07/08/2022] [Accepted: 07/20/2022] [Indexed: 11/30/2022] Open
Abstract
B cells recognize antigens via membrane‐expressed B‐cell receptors (BCR) and antibodies. Similar human BCR sequences are frequently found at a significantly higher frequency than that theoretically calculated. Patients infected with SARS‐CoV2 and HIV or with autoimmune diseases share very similar BCRs. Therefore, in silico reconstitution of BCR repertoires and identification of stereotypical BCR sequences related to human pathology have diagnostic potential. Furthermore, monitoring changes of clinically significant BCR sequences and isotype conversion has prognostic potential. For BCR repertoire analysis, peripheral blood (PB) is the most convenient source. However, the optimal human PB volume for in silico reconstitution of the BCR repertoire has not been studied in detail. Here, we sampled 5, 10, and 20 mL PB from the left arm and 40 mL PB from the right arm of two volunteers, reconstituted in silico PB BCR repertoires, and compared their composition. In both volunteers, PB sampling over 20 mL resulted in slight increases in functional unique sequences (FUSs) or almost no increase in repertoire diversity. All FUSs with a frequency above 0.08% or 0.03% in the 40 mL PB BCR repertoire were detected even in the 5 mL PB BCR repertoire from each volunteer. FUSs with a higher frequency were more likely to be found in BCR repertoires from reduced PB volume, and those coexisting in two repertoires showed a statistically significant correlation in frequency irrespective of sampled anatomical site. The correlation was more significant in higher‐frequency FUSs. These observations support the potential of BCR repertoire analysis for diagnosis.
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Affiliation(s)
- Hyunho Lee
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Korea
| | - Duck Kyun Yoo
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, 03080, Korea.,Department of Biomedical Science, Seoul National University College of Medicine, Seoul, 03080, Korea
| | - Jerome Han
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, 03080, Korea.,Department of Biomedical Science, Seoul National University College of Medicine, Seoul, 03080, Korea
| | - Ki Hyun Kim
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, 03080, Korea.,Cancer Research Institute, Seoul National University College of Medicine, Seoul, 03080, Korea
| | - Jinsung Noh
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Korea
| | - Yonghee Lee
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Korea
| | - Eunjae Lee
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, 03080, Korea.,Department of Biomedical Science, Seoul National University College of Medicine, Seoul, 03080, Korea
| | - Sunghoon Kwon
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Korea.,Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Korea.,BK21+ Creative Research Engineer Development for IT, Seoul National University, Seoul, 08826, Korea.,Bio-MAX Institute, Seoul National University, Seoul, 08826, Korea
| | - Junho Chung
- Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, 03080, Korea.,Department of Biomedical Science, Seoul National University College of Medicine, Seoul, 03080, Korea.,Cancer Research Institute, Seoul National University College of Medicine, Seoul, 03080, Korea
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Cullen JN, Martin J, Vilella AJ, Treeful A, Sargan D, Bradley A, Friedenberg SG. Development and application of a next-generation sequencing protocol and bioinformatics pipeline for the comprehensive analysis of the canine immunoglobulin repertoire. PLoS One 2022; 17:e0270710. [PMID: 35802654 PMCID: PMC9269486 DOI: 10.1371/journal.pone.0270710] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Accepted: 06/15/2022] [Indexed: 11/18/2022] Open
Abstract
Profiling the adaptive immune repertoire using next generation sequencing (NGS) has become common in human medicine, showing promise in characterizing clonal expansion of B cell clones through analysis of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IGH locus only. The objectives of this study were to: (1) describe a novel NGS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma. RNA from peripheral blood mononuclear cells of healthy dogs (n = 25) and dogs newly diagnosed with intermediate-to-large B-cell lymphoma (n = 18) with intent to pursue chemotherapy was isolated, converted into cDNA and sequenced by NGS. The BCR repertoires were identified and quantified using a novel analysis pipeline. The IGK repertoires of the healthy dogs were far less diverse compared to IGL which, as with IGH, was highly diverse. Strong biases at key positions within the CDR3 sequence were identified within the healthy dog BCR repertoire. For a subset of the dogs with B-cell lymphoma, clonal expansion of specific IGH sequences pre-treatment and reduction post-treatment was observed. The degree of expansion and reduction correlated with the clinical outcome in this subset. Future studies employing these techniques may improve disease monitoring, provide earlier recognition of disease progression, and ultimately lead to more targeted therapeutics.
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Affiliation(s)
- Jonah N. Cullen
- Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, Minnesota, United States of America
| | - Jolyon Martin
- Wellcome Trust Genome Campus, Hinxton, Saffron Walden, United Kingdom
- PetMedix Ltd, Glenn Berge Building, Babraham Research Campus, Cambridge, United Kingdom
| | - Albert J. Vilella
- PetMedix Ltd, Glenn Berge Building, Babraham Research Campus, Cambridge, United Kingdom
| | - Amy Treeful
- Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, Minnesota, United States of America
| | - David Sargan
- Department of Veterinary Medicine, Madingley Road, Cambridge, United Kingdom
| | - Allan Bradley
- Wellcome Trust Genome Campus, Hinxton, Saffron Walden, United Kingdom
- PetMedix Ltd, Glenn Berge Building, Babraham Research Campus, Cambridge, United Kingdom
- Department of Medicine, Jeffrey Cheah Biomedical Centre, Cambridge, United Kingdom
| | - Steven G. Friedenberg
- Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, Minnesota, United States of America
- * E-mail:
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40
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Chen GM, Melenhorst JJ, Tan K. B cell targeting in CAR T cell therapy: Side effect or driver of CAR T cell function? Sci Transl Med 2022; 14:eabn3353. [PMID: 35731887 DOI: 10.1126/scitranslmed.abn3353] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Chimeric antigen receptor (CAR) T cell therapies targeting CD19 and CD22 have been successful for treating B cell cancers, but CAR T cells targeting non-B cell cancers remain unsuccessful. We propose that rather than being strictly a side effect of therapy, the large number of CAR interactions with normal B cells may be a key contributor to clinical CAR T cell responses.
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Affiliation(s)
- Gregory M Chen
- Graduate Group in Genomics and Computational Biology, University of Pennsylvania, Philadelphia, PA, USA
| | - Jan Joseph Melenhorst
- Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA
| | - Kai Tan
- Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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41
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Lupo C, Spisak N, Walczak AM, Mora T. Learning the statistics and landscape of somatic mutation-induced insertions and deletions in antibodies. PLoS Comput Biol 2022; 18:e1010167. [PMID: 35653375 PMCID: PMC9197026 DOI: 10.1371/journal.pcbi.1010167] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 06/14/2022] [Accepted: 05/05/2022] [Indexed: 11/25/2022] Open
Abstract
Affinity maturation is crucial for improving the binding affinity of antibodies to antigens. This process is mainly driven by point substitutions caused by somatic hypermutations of the immunoglobulin gene. It also includes deletions and insertions of genomic material known as indels. While the landscape of point substitutions has been extensively studied, a detailed statistical description of indels is still lacking. Here we present a probabilistic inference tool to learn the statistics of indels from repertoire sequencing data, which overcomes the pitfalls and biases of standard annotation methods. The model includes antibody-specific maturation ages to account for variable mutational loads in the repertoire. After validation on synthetic data, we applied our tool to a large dataset of human immunoglobulin heavy chains. The inferred model allows us to identify universal statistical features of indels in heavy chains. We report distinct insertion and deletion hotspots, and show that the distribution of lengths of indels follows a geometric distribution, which puts constraints on future mechanistic models of the hypermutation process. Affinity maturation of B cell receptors is an important mechanism by which our body designs neutralizing antibodies to defend us against pathogens, including broadly neutralizing antibodies, which target a wide range of variants of the same pathogen. Such antibodies often contain key insertions and deletions to the germline gene, or “indels”, which are caused by somatic hypermutations. However, the mechanism, frequency and role of these indels are still elusive. We designed a computational method based on a probabilistic framework to infer the characteristics of this mutational process from high-throughput antibody sequencing experiments. Applied to human data, our approach provides a comprehensive quantitative description of insertions and deletions, opening avenues for better understanding the process of affinity maturation and the design of vaccines for eliciting a broad antibody response.
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Affiliation(s)
- Cosimo Lupo
- Laboratoire de physique de l’École normale supérieure, CNRS, PSL University, Sorbonne Université, and Université de Paris, Paris, France
| | - Natanael Spisak
- Laboratoire de physique de l’École normale supérieure, CNRS, PSL University, Sorbonne Université, and Université de Paris, Paris, France
| | - Aleksandra M. Walczak
- Laboratoire de physique de l’École normale supérieure, CNRS, PSL University, Sorbonne Université, and Université de Paris, Paris, France
- * E-mail: (AMW); (TM)
| | - Thierry Mora
- Laboratoire de physique de l’École normale supérieure, CNRS, PSL University, Sorbonne Université, and Université de Paris, Paris, France
- * E-mail: (AMW); (TM)
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42
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Kanduri C, Pavlović M, Scheffer L, Motwani K, Chernigovskaya M, Greiff V, Sandve GK. Profiling the baseline performance and limits of machine learning models for adaptive immune receptor repertoire classification. Gigascience 2022; 11:giac046. [PMID: 35639633 PMCID: PMC9154052 DOI: 10.1093/gigascience/giac046] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Revised: 12/23/2021] [Accepted: 04/08/2022] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Machine learning (ML) methodology development for the classification of immune states in adaptive immune receptor repertoires (AIRRs) has seen a recent surge of interest. However, so far, there does not exist a systematic evaluation of scenarios where classical ML methods (such as penalized logistic regression) already perform adequately for AIRR classification. This hinders investigative reorientation to those scenarios where method development of more sophisticated ML approaches may be required. RESULTS To identify those scenarios where a baseline ML method is able to perform well for AIRR classification, we generated a collection of synthetic AIRR benchmark data sets encompassing a wide range of data set architecture-associated and immune state-associated sequence patterns (signal) complexity. We trained ≈1,700 ML models with varying assumptions regarding immune signal on ≈1,000 data sets with a total of ≈250,000 AIRRs containing ≈46 billion TCRβ CDR3 amino acid sequences, thereby surpassing the sample sizes of current state-of-the-art AIRR-ML setups by two orders of magnitude. We found that L1-penalized logistic regression achieved high prediction accuracy even when the immune signal occurs only in 1 out of 50,000 AIR sequences. CONCLUSIONS We provide a reference benchmark to guide new AIRR-ML classification methodology by (i) identifying those scenarios characterized by immune signal and data set complexity, where baseline methods already achieve high prediction accuracy, and (ii) facilitating realistic expectations of the performance of AIRR-ML models given training data set properties and assumptions. Our study serves as a template for defining specialized AIRR benchmark data sets for comprehensive benchmarking of AIRR-ML methods.
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Affiliation(s)
- Chakravarthi Kanduri
- Centre for Bioinformatics, Department of Informatics, University of Oslo, Oslo 0373, Norway
| | - Milena Pavlović
- Centre for Bioinformatics, Department of Informatics, University of Oslo, Oslo 0373, Norway
| | - Lonneke Scheffer
- Centre for Bioinformatics, Department of Informatics, University of Oslo, Oslo 0373, Norway
| | - Keshav Motwani
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida,
FL 32610, USA
| | - Maria Chernigovskaya
- Department of Immunology and Oslo University Hospital, University of Oslo, Oslo, 0372, Norway
| | - Victor Greiff
- Department of Immunology and Oslo University Hospital, University of Oslo, Oslo, 0372, Norway
| | - Geir K Sandve
- Centre for Bioinformatics, Department of Informatics, University of Oslo, Oslo 0373, Norway
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43
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Ledsgaard L, Ljungars A, Rimbault C, Sørensen CV, Tulika T, Wade J, Wouters Y, McCafferty J, Laustsen AH. Advances in antibody phage display technology. Drug Discov Today 2022; 27:2151-2169. [PMID: 35550436 DOI: 10.1016/j.drudis.2022.05.002] [Citation(s) in RCA: 80] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Revised: 03/24/2022] [Accepted: 05/04/2022] [Indexed: 01/06/2023]
Abstract
Phage display technology can be used for the discovery of antibodies for research, diagnostic, and therapeutic purposes. In this review, we present and discuss key parameters that can be optimized when performing phage display selection campaigns, including the use of different antibody formats and advanced strategies for antigen presentation, such as immobilization, liposomes, nanodiscs, virus-like particles, and whole cells. Furthermore, we provide insights into selection strategies that can be used for the discovery of antibodies with complex binding requirements, such as targeting a specific epitope, cross-reactivity, or pH-dependent binding. Lastly, we provide a description of specialized phage display libraries for the discovery of bispecific antibodies and pH-sensitive antibodies. Together, these methods can be used to improve antibody discovery campaigns against all types of antigen. Teaser: This review provides an overview of the different strategies that can be exploited to improve the success rate of antibody phage display discovery campaigns, addressing key parameters, such as antigen presentation, selection methodologies, and specialized libraries.
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Affiliation(s)
- Line Ledsgaard
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark.
| | - Anne Ljungars
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - Charlotte Rimbault
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - Christoffer V Sørensen
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - Tulika Tulika
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - Jack Wade
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - Yessica Wouters
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
| | - John McCafferty
- Department of Medicine, Addenbrookes Hospital, Box 157, Hills Road, Cambridge, CB2 0QQ, UK; Department of Medicine, Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK
| | - Andreas H Laustsen
- Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark.
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44
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Sun Z, Li W, Mellors JW, Orentas R, Dimitrov DS. Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22. Front Immunol 2022; 13:869825. [PMID: 35464476 PMCID: PMC9019674 DOI: 10.3389/fimmu.2022.869825] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2022] [Accepted: 03/16/2022] [Indexed: 12/03/2022] Open
Abstract
Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.
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Affiliation(s)
- Zehua Sun
- Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, Pittsburgh, PA, United States
| | - Wei Li
- Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, Pittsburgh, PA, United States
| | - John W. Mellors
- Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, Pittsburgh, PA, United States
- Abound Bio, Pittsburgh, PA, United States
| | - Rimas Orentas
- Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, United States
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, United States
| | - Dimiter S. Dimitrov
- Center for Antibody Therapeutics, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh Medical School, Pittsburgh, PA, United States
- Abound Bio, Pittsburgh, PA, United States
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45
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Kenny SE, Antaw F, Locke WJ, Howard CB, Korbie D, Trau M. Next-Generation Molecular Discovery: From Bottom-Up In Vivo and In Vitro Approaches to In Silico Top-Down Approaches for Therapeutics Neogenesis. Life (Basel) 2022; 12:363. [PMID: 35330114 PMCID: PMC8950575 DOI: 10.3390/life12030363] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Accepted: 02/23/2022] [Indexed: 12/02/2022] Open
Abstract
Protein and drug engineering comprises a major part of the medical and research industries, and yet approaches to discovering and understanding therapeutic molecular interactions in biological systems rely on trial and error. The general approach to molecular discovery involves screening large libraries of compounds, proteins, or antibodies, or in vivo antibody generation, which could be considered "bottom-up" approaches to therapeutic discovery. In these bottom-up approaches, a minimal amount is known about the therapeutics at the start of the process, but through meticulous and exhaustive laboratory work, the molecule is characterised in detail. In contrast, the advent of "big data" and access to extensive online databases and machine learning technologies offers promising new avenues to understanding molecular interactions. Artificial intelligence (AI) now has the potential to predict protein structure at an unprecedented accuracy using only the genetic sequence. This predictive approach to characterising molecular structure-when accompanied by high-quality experimental data for model training-has the capacity to invert the process of molecular discovery and characterisation. The process has potential to be transformed into a top-down approach, where new molecules can be designed directly based on the structure of a target and the desired function, rather than performing screening of large libraries of molecular variants. This paper will provide a brief evaluation of bottom-up approaches to discovering and characterising biological molecules and will discuss recent advances towards developing top-down approaches and the prospects of this.
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Affiliation(s)
- Sophie E. Kenny
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner of College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia; (S.E.K.); (F.A.); (C.B.H.)
| | - Fiach Antaw
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner of College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia; (S.E.K.); (F.A.); (C.B.H.)
| | - Warwick J. Locke
- Molecular Diagnostic Solutions, Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Building 101, Clunies Ross Street, Canberra, ACT 2601, Australia;
| | - Christopher B. Howard
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner of College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia; (S.E.K.); (F.A.); (C.B.H.)
| | - Darren Korbie
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner of College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia; (S.E.K.); (F.A.); (C.B.H.)
| | - Matt Trau
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner of College and Cooper Roads (Bldg 75), Brisbane, QLD 4072, Australia; (S.E.K.); (F.A.); (C.B.H.)
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
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46
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Hom JR, Tomar D, Tipton CM. Exploring the Diversity of the B-Cell Receptor Repertoire Through High-Throughput Sequencing. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2022; 2421:231-241. [PMID: 34870823 DOI: 10.1007/978-1-0716-1944-5_16] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Repertoire sequencing of B cells is the high-throughput profiling of B cell receptors (BCR) expressed on the surface of B cells and of immunoglobulins (Ig) expressed by antibody secreting cells. Each BCR/Ig transcript has a unique complementarity-determining region 3 (CDR3) sequence that can be used to identify and track individual B cell lymphocytes over time and throughout different compartments of the human body. B cell differentiation can be further tracked by assessing the point mutations acquired during affinity maturation via somatic hypermutation (SHM). Here we describe a method for high-throughput sequencing of the variable region of Ig heavy-chain transcripts for repertoire analysis of human B cells on the Illumina Miseq platform.
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Affiliation(s)
- Jennifer R Hom
- Lowance Center for Human Immunology, Division of Rheumatology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Deepak Tomar
- Lowance Center for Human Immunology, Division of Rheumatology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
- Department of Medicine, Division of Rheumatology, Emory University, Atlanta, GA, USA
| | - Christopher M Tipton
- Lowance Center for Human Immunology, Division of Rheumatology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA.
- Department of Medicine, Division of Rheumatology, Emory University, Atlanta, GA, USA.
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47
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Ferrall-Fairbanks MC, Chakiryan N, Chobrutskiy BI, Kim Y, Teer JK, Berglund A, Mulé JJ, Fournier M, Siegel EM, Dhillon J, Falasiri SSA, Arturo JF, Katende EN, Blanck G, Manley BJ, Altrock PM. Quantification of T- and B-cell immune receptor distribution diversity characterizes immune cell infiltration and lymphocyte heterogeneity in clear cell renal cell carcinoma. Cancer Res 2022; 82:929-942. [DOI: 10.1158/0008-5472.can-21-1747] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 11/02/2021] [Accepted: 01/10/2022] [Indexed: 11/16/2022]
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48
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James CA, Xu Y, Aguilar MS, Jing L, Layton ED, Gilleron M, Minnaard AJ, Scriba TJ, Day CL, Warren EH, Koelle DM, Seshadri C. CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells. Nat Commun 2022; 13:78. [PMID: 35013257 PMCID: PMC8748927 DOI: 10.1038/s41467-021-27764-w] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Accepted: 12/10/2021] [Indexed: 12/13/2022] Open
Abstract
T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.
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Affiliation(s)
- Charlotte A James
- Molecular Medicine and Mechanisms of Disease PhD Program (M3D), Department of Pathology, University of Washington, Seattle, WA, USA
| | - Yuexin Xu
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
| | | | - Lichen Jing
- Department of Medicine, University of Washington, Seattle, WA, USA
| | - Erik D Layton
- Department of Medicine, University of Washington, Seattle, WA, USA
| | - Martine Gilleron
- Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077, Toulouse, France
| | - Adriaan J Minnaard
- Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands
| | - Thomas J Scriba
- South African Tuberculosis Vaccine Initiative and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, Department of Pathology, University of Cape Town, Cape Town, South Africa
| | - Cheryl L Day
- Emory Vaccine Center and Department of Microbiology and Immunology, Emory University, Atlanta, GA, USA
| | - Edus H Warren
- Molecular Medicine and Mechanisms of Disease PhD Program (M3D), Department of Pathology, University of Washington, Seattle, WA, USA
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Department of Medicine, University of Washington, Seattle, WA, USA
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
| | - David M Koelle
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Department of Medicine, University of Washington, Seattle, WA, USA
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
- Department of Global Health, University of Washington, Seattle, WA, USA
- Benaroya Research Institute, Seattle, WA, USA
| | - Chetan Seshadri
- Department of Medicine, University of Washington, Seattle, WA, USA.
- Tuberculosis Research and Training Center, Seattle, WA, USA.
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49
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Davydova EK. Protein Engineering: Advances in Phage Display for Basic Science and Medical Research. BIOCHEMISTRY. BIOKHIMIIA 2022; 87:S146-S110. [PMID: 35501993 PMCID: PMC8802281 DOI: 10.1134/s0006297922140127] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Revised: 10/28/2021] [Accepted: 11/02/2021] [Indexed: 12/03/2022]
Abstract
Functional Protein Engineering became the hallmark in biomolecule manipulation in the new millennium, building on and surpassing the underlying structural DNA manipulation and recombination techniques developed and employed in the last decades of 20th century. Because of their prominence in almost all biological processes, proteins represent extremely important targets for engineering enhanced or altered properties that can lead to improvements exploitable in healthcare, medicine, research, biotechnology, and industry. Synthetic protein structures and functions can now be designed on a computer and/or evolved using molecular display or directed evolution methods in the laboratory. This review will focus on the recent trends in protein engineering and the impact of this technology on recent progress in science, cancer- and immunotherapies, with the emphasis on the current achievements in basic protein research using synthetic antibody (sABs) produced by phage display pipeline in the Kossiakoff laboratory at the University of Chicago (KossLab). Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.
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Affiliation(s)
- Elena K Davydova
- The University of Chicago, Department of Biochemistry and Molecular Biology, Chicago, IL 60637, USA.
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50
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Abstract
The development of high-throughput sequencing of adaptive immune receptor repertoires (AIRR-seq of IG and TR rearrangements) has provided a new frontier for in-depth analysis of the immune system. The last decade has witnessed an explosion in protocols, experimental methodologies, and computational tools. In this chapter, we discuss the major considerations in planning a successful AIRR-seq experiment together with basic strategies for controlling and evaluating the outcome of the experiment. Members of the AIRR Community have authored several chapters in this edition, which cover step-by-step instructions to successfully conduct, analyze, and share an AIRR-seq project.
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