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1. Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice

Author

Andrew C. Hunt, James Brett Case, Young-Jun Park, Longxing Cao, Kejia Wu, Alexandra C. Walls, Zhuoming Liu, John E. Bowen, Hsien-Wei Yeh, Shally Saini, Louisa Helms, Yan Ting Zhao, Tien-Ying Hsiang, Tyler N. Starr, Inna Goreshnik, Lisa Kozodoy, Lauren Carter, Rashmi Ravichandran, Lydia B. Green, Wadim L. Matochko, Christy A. Thomson, Bastian Vögeli, Antje Krüger, Laura A. VanBlargan, Rita E. Chen, Baoling Ying, Adam L. Bailey, Natasha M. Kafai, Scott E. Boyken, Ajasja Ljubetič, Natasha Edman, George Ueda, Cameron M. Chow, Max Johnson, Amin Addetia, Mary-Jane Navarro, Nuttada Panpradist, Michael Gale, Benjamin S. Freedman, Jesse D. Bloom, Hannele Ruohola-Baker, Sean P. J. Whelan, Lance Stewart, Michael S. Diamond, David Veesler, Michael C. Jewett, and David Baker

Subjects
Autoimmune Disease, Medical and Health Sciences, Antibodies, Vaccine Related, Mice, Biodefense, Animals, Humans, Viral, Neutralizing, Lung, SARS-CoV-2, Prevention, Cryoelectron Microscopy, COVID-19, General Medicine, Pneumonia, Biological Sciences, Spike Glycoprotein, Coronavirus, Emerging Infectious Diseases, Infectious Diseases, 5.1 Pharmaceuticals, Pneumonia & Influenza, Development of treatments and therapeutic interventions, Biotechnology
Abstract

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Published
2022
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2. Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Author

David Veesler, James Brett Case, Sean P. J. Whelan, Benjamin S. Freedman, Hsien-Wei Yeh, Ajasja Ljubetič, Hannele Ruohola-Baker, Barry R. Lutz, Louisa Helms, Bastain Vogeli, Yan Ting Zhao, Laura A. VanBlargan, Baoling Ying, David Baker, Natasha I. Edman, Jesse D. Bloom, Natasha M. Kafai, Antje Kruger-gericke, Lauren Carter, Adam L. Bailey, Cameron M. Chow, John E. Bowen, George Ueda, Shally Saini, Amin Addetia, Inna Goreshnik, Michael Gale, Longxing Cao, Scott E. Boyken, Rita E. Chen, Andrew Hunt, Lisa Kozodoy, Kejia Wu, Michael S. Diamond, Wadim L. Matochko, Tien-Ying Hsiang, Michael C. Jewett, Young-Jun Park, Alexandra C. Walls, Lance Stewart, Rashmi Ravichandran, Zhuoming Liu, Christy Ann Thomson, Tyler N. Starr, Nuttada Panpradist, and Lydia Green

Subjects
2019-20 coronavirus outbreak, Coronavirus disease 2019 (COVID-19), SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Pandemic preparedness, Cryoelectron Microscopy, COVID-19, Computational biology, Biology, Antibodies, Viral, Antibodies, Neutralizing, Article, Mice, Spike Glycoprotein, Coronavirus, Ic50 values, Animals, Humans
Abstract

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness., One-Sentence Summary: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

Published
2021

3. Pandemic H1N1 influenza infection and vaccination in humans induces cross-protective antibodies that target the hemagglutinin stem

Author

Christy Ann Thomson, Yanni eWang, Linda eJackson, Melanie eOlson, Welson eWang, Alena eLiavonchanka, Liya eKeleta, Vanessa eSilva, Sandra eDiederich, R Brad Jones, Jonathan eGubbay, John ePasick, Martin ePetric, Francois eJean, Vanessa eAllen, Earl eBrown, James eRini, and John William Schrader

Subjects
Vaccines, competition for T cell help, cross-protective antibodies, hemagglutinin, heterosubtypic, memory B cells, Immunologic diseases. Allergy, RC581-607
Abstract

Most monoclonal antibodies (mAbs) generated from humans infected or vaccinated with the 2009 pandemic H1N1 (pdmH1N1) influenza virus targeted the hemagglutinin (HA) stem. These anti-HA stem mAbs mostly used IGHV1-69 and bound readily to epitopes on the conventional seasonal influenza and pdmH1N1 vaccines. The anti-HA stem mAbs neutralized pdmH1N1, seasonal influenza H1N1 and avian H5N1 influenza viruses by inhibiting HA-mediated fusion of membranes and protected against and treated heterologous lethal infections in mice with H5N1 influenza virus. This demonstrated that therapeutic mAbs could be generated a few months after the new virus emerged. Human immunization with the pdmH1N1 vaccine induced circulating antibodies that protected mice from lethal, heterologous H5N1 influenza infections. We observed that the dominant heterosubtypic antibody response against the HA stem correlated with the relative absence of memory B cells against the HA head of pdmH1N1, thus enabling the rare heterosubtypic memory B cells induced by seasonal influenza and specific for conserved sites on the HA stem to compete for T-cell help. These results support the notion that broadly protective antibodies against influenza would be induced by successive vaccination with conventional influenza vaccines based on subtypes of HA in viruses not circulating in humans.

Published
2012
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4. A simplified method for the efficient refolding and purification of recombinant human GM-CSF.

Author

Christy A Thomson, Melanie Olson, Linda M Jackson, and John W Schrader

Subjects
Medicine, Science
Abstract

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.

Published
2012
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5. Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins

Author

Christy Ann Thomson, Bernd Gardill, Lawrence Tu, Jerry Huang, Filip Van Petegem, and Kirill Oxenoid

Subjects
Models, Molecular, 0301 basic medicine, Scaffold protein, Protein Conformation, medicine.drug_class, Recombinant Fusion Proteins, Protein domain, lcsh:Medicine, Monoclonal antibody, Article, Flow cytometry, Antigen-Antibody Reactions, Mice, 03 medical and health sciences, Drug Delivery Systems, 0302 clinical medicine, Protein structure, Bacterial Proteins, Protein Domains, Antibody Specificity, medicine, Animals, lcsh:Science, Nanodisc, Arcobacter, Hybridomas, Multidisciplinary, medicine.diagnostic_test, Chemistry, NAV1.7 Voltage-Gated Sodium Channel, lcsh:R, Antibodies, Monoclonal, Membrane Proteins, Flow Cytometry, Nanostructures, Cell biology, 030104 developmental biology, Membrane, Solubility, Membrane protein, Immunoglobulin G, Ion channels, lcsh:Q, Antibody therapy, 030217 neurology & neurosurgery
Abstract

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.

Published
2020
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6. The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities

Author

Emil F. Pai, Steve Bryson, Urmi Dhagat, Michael W. Parker, Christy A. Thomson, Sophie E. Broughton, John W. Schrader, Angel F. Lopez, Emma F Barry, Barbara J. McClure, Tracy L. Nero, Timothy R. Hercus, Karen S. Cheung Tung Shing, Dhagat, Urmi, Hercus, Timothy R, Broughton, Sophie E, Nero, Tracy L, Cheung Tung Shing, Karen S, Barry, Emma F, Thomson, Christy A, Bryson, Steve, Pai, Emil F, McClure, Barbara J, Schrader, John W, Lopez, Angel F, and Parker, Michael W

Subjects
0301 basic medicine, medicine.drug_class, Protein Conformation, Beta common cytokines, pulmonary alveolar proteinosis, Hematopoietic growth factor, medicine.medical_treatment, Immunology, Antigen-Antibody Complex, Monoclonal antibody, Crystallography, X-Ray, Epitope, Arthritis, Rheumatoid, 03 medical and health sciences, Epitopes, 0302 clinical medicine, Immune system, Report, medicine, Immunology and Allergy, Humans, X-ray crystallography, Autoantibodies, Auto-antibodies, biology, Molecular Structure, Chemistry, Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF, Colony-stimulating factor, Antibodies, Neutralizing, 030104 developmental biology, Granulocyte macrophage colony-stimulating factor, Cytokine, 030220 oncology & carcinogenesis, biology.protein, Cancer research, Cytokines, anti-GM-CSF therapeutics, Immunotherapy, Antibody, medicine.drug, Protein Binding
Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders Refereed/Peer-reviewed

Published
2018

7. Characterization of pathogenic human monoclonal autoantibodies against GM-CSF

Author

Gary P. Anderson, Thomas K. Waddell, Melanie Olson, Timothy R. Hercus, Tracy L. Nero, John W. Schrader, Yanni Wang, Michael W. Parker, Lenka Allan, John A. Hamilton, Christy A. Thomson, Angel L. Lopez, Amanda L Turner, and Linda M. Jackson

Subjects
Neutrophils, T-Lymphocytes, Pulmonary Alveolar Proteinosis, Epitope, Inhibitory Concentration 50, Cell Line, Tumor, medicine, Humans, Point Mutation, Autoantibodies, Cell Proliferation, B-Lymphocytes, CD11b Antigen, Multidisciplinary, biology, business.industry, Autoantibody, Antibodies, Monoclonal, Granulocyte-Macrophage Colony-Stimulating Factor, Surface Plasmon Resonance, Biological Sciences, medicine.disease, Anti-thyroid autoantibodies, Kinetics, Epitope mapping, Mutation, Monoclonal, Immunology, biology.protein, Antibody, Pulmonary alveolar proteinosis, business, Immunologic Memory, Epitope Mapping, Anti-SSA/Ro autoantibodies
Abstract

The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.

Published
2013
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8. Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies

Author

Brian Mingtung Chan, Robert Y.-T. Chou, Ognjen Grujic, Stephanie A Grauer, Kevin Graham, Christy Ann Thomson, Yang Li, Jennifer Weiszmann, Laura Sekirov, Jennitte Stevens, Ian Foltz, Kathy Manchulenko, Anita Badh, and Thomas M. Dillon

Subjects
0301 basic medicine, Agonist, medicine.drug_class, Biophysics, Receptors, Cell Surface, CHO Cells, Monoclonal antibody, Biochemistry, 03 medical and health sciences, Structure-Activity Relationship, 0302 clinical medicine, Cricetulus, Cell surface receptor, Orexin Receptors, parasitic diseases, medicine, Animals, Protein Isoforms, Avidity, Disulfides, Receptor, Molecular Biology, Klotho Proteins, biology, Antibodies, Monoclonal, Membrane Proteins, Biological activity, Cell Biology, Primary and secondary antibodies, Molecular biology, 030104 developmental biology, 030220 oncology & carcinogenesis, Antigens, Surface, biology.protein, Antibody, Epitope Mapping
Abstract

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Published
2017

9. Somatic Diversity in CDR3 Loops Allows Single V-Genes To Encode Innate Immunological Memories for Multiple Pathogens

Author

Christy A. Thomson, Ken Q. Little, Donald C. Reason, and John W. Schrader

Subjects
Glycan, Neutrophils, Somatic cell, Protein subunit, Molecular Sequence Data, Immunology, Immunoglobulin Variable Region, Biology, medicine.disease_cause, DNA sequencing, Germline, Streptococcus pneumoniae, medicine, Humans, Immunology and Allergy, Amino Acid Sequence, Binding site, Cells, Cultured, Genetics, chemistry.chemical_classification, Antibodies, Monoclonal, Complementarity Determining Regions, Molecular biology, Immunity, Innate, Respiratory Syncytial Viruses, Amino acid, chemistry, biology.protein, Immunoglobulin Light Chains, Binding Sites, Antibody, Somatic Hypermutation, Immunoglobulin, Immunoglobulin Heavy Chains, Immunologic Memory
Abstract

The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized l-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.

Published
2011
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10. Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders

Author

Giuseppe F. Pontoriero, Kevin Skoblenick, Christy A. Thomson, Zdenek B. Pristupa, Joseph Gabriele, Ram K. Mishra, and Nancy Thomas

Subjects
Dopamine, RNA Splicing, Molecular Sequence Data, Nerve Tissue Proteins, Biology, Biochemistry, Mice, Central Nervous System Diseases, Cell Line, Tumor, Sequence Homology, Nucleic Acid, Complementary DNA, Heat shock protein, Animals, Humans, HSP70 Heat-Shock Proteins, Amino Acid Sequence, Cloning, Molecular, Peptide sequence, Neurons, Dopamine binding, Original Paper, Messenger RNA, Base Sequence, Alternative splicing, Nucleic acid sequence, Cell Biology, Molecular biology, Recombinant Proteins, Catecholamine binding, Haloperidol, Cattle
Abstract

Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed chaperone activity. Radiolabeled dopamine binding studies with recombinant human CRP40 protein demonstrated the ability of this protein to bind dopamine with low affinity and high capacity. The full-length human CRP40 nucleotide sequence was elucidated (accession #DQ480334) with RNA ligase-mediated rapid amplification of complementary DNA ends polymerase chain reaction, while Northern blot hybridization suggested that human CRP40 is an alternative splice variant of the 70-kDa mitochondrial heat shock protein, mortalin. Human SH-SY5Y neuroblastoma cells treated with the antipsychotic drug, haloperidol, exhibited a significant increase in CRP40 messenger RNA expression compared to untreated control cells, while other dopamine agonists/antagonists also altered CRP40 expression and immunolocalization. In conclusion, these results show that we have cloned a splice variant of mortalin with a novel catecholamine binding function and that this chaperone-like protein may be neuroprotective in dopamine-related central nervous system disorders.

Published
2009
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11. IgG Structure and Function

Author

Christy Ann Thomson

Subjects
chemistry.chemical_classification, Effector, Immunoglobulin domain, Biology, Immunoglobulin light chain, Molecular biology, Immunoglobulin G, Cell biology, chemistry, Antigen, Humoral immunity, biology.protein, Antibody, Glycoprotein
Abstract

Immunoglobulin G (IgG) is a 150 kDa glycoprotein found in human serum and an important mediator of the humoral immune response. IgG is composed of two heavy chains and two light chains, each divided into variable and constant domains. The variable domains are responsible for antigen binding, while the constant domains are responsible for interactions with effector molecules and effector cells. Human IgG is divided into four subclasses which although having high sequence homology, offer structural variation that significantly impacts biological function.

Published
2016
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12. Identification of Small Molecule Chemical Inhibitors of the Collagen-Specific Chaperone Hsp47

Author

Vettai S. Ananthanarayanan, Helen M. Atkinson, and Christy A. Thomson

Subjects
chemistry.chemical_classification, animal structures, biology, Chemistry, Nitro compound, Aromatic amine, Small molecule, In vitro, Pathogenesis, Biochemistry, Chaperone (protein), Heat shock protein, embryonic structures, Drug Discovery, biology.protein, Molecular Medicine, Glycoprotein
Abstract

Hsp47 is a collagen-specific molecular chaperone whose activity has been implicated in the pathogenesis of fibrotic diseases. Here, we describe the development of an assay for screening libraries of chemical compounds for inhibitors of Hsp47. A preliminary screen of 2080 compounds identified four that demonstrated inhibitory activity against Hsp47 in vitro, with IC(50) values ranging from 3 to 27 muM. Compounds identified through this method may provide the basis for development of novel antifibrotic therapeutics.

Published
2005
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13. Structures of Preferred Human IgV Genes-Based Protective Antibodies Identify How Conserved Residues Contact Diverse Antigens and Assign Source of Specificity to CDR3 Loop Variation

Author

Emil F. Pai, Kenneth J. Smith, Somnath Dasgupta, John W. Schrader, Christy A. Thomson, Louise Fremgaard Risnes, and Steve Bryson

Subjects
0301 basic medicine, Serotype, Protein Conformation, Immunology, Immunoglobulin Variable Region, Cytomegalovirus, Peptide, Biology, 03 medical and health sciences, Immunoglobulin Fab Fragments, 0302 clinical medicine, Protein structure, Antigen, Immunology and Allergy, Humans, Antigens, Gene, chemistry.chemical_classification, Genetic Variation, Evolutionary pressure, Amino acid, 030104 developmental biology, Streptococcus pneumoniae, Membrane protein, Biochemistry, chemistry, 030215 immunology
Abstract

The human Ab response to certain pathogens is oligoclonal, with preferred IgV genes being used more frequently than others. A pair of such preferred genes, IGVK3-11 and IGVH3-30, contributes to the generation of protective Abs directed against the 23F serotype of the pneumonococcal capsular polysaccharide of Streptococcus pneumoniae and against the AD-2S1 peptide of the gB membrane protein of human CMV. Structural analyses of Fab fragments of mAbs 023.102 and pn132p2C05 in complex with portions of the 23F polysaccharide revealed five germline-encoded residues in contact with the key component, l-rhamnose. In the case of the AD-2S1 peptide, the KE5 Fab fragment complex identified nine germline-encoded contact residues. Two of these germline-encoded residues, Arg91L and Trp94L, contact both the l-rhamnose and the AD-2S1 peptide. Comparison of the respective paratopes that bind to carbohydrate and protein reveals that stochastic diversity in both CDR3 loops alone almost exclusively accounts for their divergent specificity. Combined evolutionary pressure by human CMV and the 23F serotype of S. pneumoniae acted on the IGVK3-11 and IGVH3-30 genes as demonstrated by the multiple germline-encoded amino acids that contact both l-rhamnose and AD-2S1 peptide.

Published
2014

14. Contributory presentations/posters

Author

N. Manoj, V. R. Srinivas, A. Surolia, M. Vijayan, K. Suguna, R. Ravishankar, R. Schwarzenbacher, K. Zeth, null Diederichs, G. M. Kostner, A. Gries, P. Laggner, R. Prassl, null Madhusudan, Pearl Akamine, Nguyen-huu Xuong, Susan S. Taylor, M. Bidva Sagar, K. Saikrishnan, S. Roy, K. Purnapatre, P. Handa, U. Varshney, B. K. Biswal, N. Sukumar, J. K. Mohana Rao, A. Johnson, Vasantha Pattabhi, S. Sri Krishna, Mira Sastri, H. S. Savithri, M. R. N. Murthy, Bindu Pillai, null Kannan, M. V. Hosur, Mukesh Kumar, Swati Patwardhan, K. K. Kannan, B. Padmanabhaa, S. Sasaki-Sugio, M. Nukaga, T. Matsuzaki, S. Karthikevan, S. Sharma, A. K. Sharma, M. Paramasivam, P. Kumar, J. A. Khan, S. Yadav, A. Srinivasan, T. P. Singh, S. Gourinath, Neelima Alam, A. Srintvasan, Vikas Chandra, Punit Kaur, Ch. Betzel, S. Ghosh, A. K. Bera, S. Bhattacharya, S. Chakraborty, A. K. Pal, B. P. Mukhopadhyay, I. Dey, U. Haldar, Asok Baneriee, Jozef Sevcik, Adriana Solovicova, K. Sekar, M. Sundaralingam, N. Genov, Dong-cai Liang, Tao Jiang, Ji-ping Zhang, Wen-rui Chang, Wolfgang Jahnke, Marcel Blommers, S. C. Panchal, R. V. Hosur, Bindu Pillay, Puniti Mathur, S. Srivatsun, Ratan Mani Joshi, N. R. Jaganathan, V. S. Chauhan, H. S. Atreya, S. C. Sahu, K. V. R. Chary, Girjesh Govil, Elisabeth Adjadj, Éric Quinjou, Nadia Izadi-Pruneyre, Yves Blouquit, Joël Mispelter, Bernadette Heyd, Guilhem Lerat, Philippe Milnard, Michel Desmadreil, Y. Lin, B. D. Nageswara Rao, Vidva Raghunathan, Mei H. Chau, Prashant Pesais, Sudha Srivastava, Evans Coutinho, Anil Saran, Leizl F. Sapico, Jayson Gesme, Herbert Lijima, Raymond Paxton, Thamarapu Srikrishnan, C. R. Grace, G. Nagenagowda, A. M. Lynn, Sudha M. Cowsik, Sarata C. Sahu, S. Chauhan, A. Bhattacharya, G. Govil, Anil Kumar, Maurizio Pellecchia, Erik R. P. Zuiderweg, Keiichi Kawano, Tomoyasu Aizawa, Naoki Fujitani, Yoichi Hayakawa, Atsushi Ohnishi, Tadayasu Ohkubo, Yasuhiro Kumaki, Kunio Hikichi, Katsutoshi Nitta, V. Rani Parvathy, R. M. Kini, Takumi Koshiba, Yoshihiro Kobashigawa, Min Yao, Makoto Demura, Astushi Nakagawa, Isao Tanaka, Kunihiro Kuwajima, Jens Linge, Seán O. Donoghue, Michael Nilges, G. Chakshusmathi, Girish S. Ratnaparkhi, P. K. Madhu, R. Varadarajan, C. Tetreau, M. Tourbez, D. Lavalette, M. Manno, P. L. San Biagio, V. Martorana, A. Emanuele, S. M. Vaiana, D. Bulone, M. B. Palma-Vittorelli, M. U. Palma, V. D. Trivedi, S. F. Cheng, W. J. Chien, S. H. Yang, S. Francis, D. K. Chang, Renn Batra, Michael A. Geeves, Dietmar J. Manstein, Joanna Trvlska, Pawel Grochowski, Maciej Geller, K. Ginalski, P. Grochowski, B. Lesyng, P. Lavalette, Y. Blouquit, D. Roccatano, A. Amadei, A. Di Nola, H. J. C. Berendsen, Bosco Ho, P. M. G. Curmi, H. Berry, D. Lairez, E. Pauthe, J. Pelta, V. Kothekar, Shakti Sahi, M. Srinivasan, Anil K. Singh, Kartha S. Madhusudnan, Fateh S. Nandel, Harpreet Kaur, Balwinder Singh, D. V. S. Jain, K. Anton Feenstra, Herman J. C. Berendsen, F. Tama, Y. -H. Sanejouand, N. 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Bernard, Ana Damjanović, Thorsten Ritz, Klaus Schulten, Wang Jushuo, Shan Jixiu, Gong Yandao, Kuang Tingyun, Zhao Nanming, Arvi Freiberg, Kõu Timpmann, Rein Ruus, Neal W. Woodbury, E. V. Nemtseva, N. S. Kudryasheva, A. G. Sizykh, V. N. Shikhov, T. V. Nesterenko, A. A. Tikhomirov, Giorgio Forti, Giovanni Finazzi, Alberto Furia, Romina Paola Barbagallo, S. Iskenderova, R. Agalarov, R. Gasanov, Miyashita Osamu, G. O. Nobuhiro, R. K. Soni, M. Ramrakhiani, Hiromasa Yagi, Kacko Tozawa, Nobuaki Sekino, Tomoyuki Iwabuchi, Masasuke Yoshida, Hideo Akutsu, A. V. Avetisyan, A. D. Kaulen, V. P. Skulachev, B. A. Feniouk, Cécile Breyton, Werner Kühlbrandt, Maria Assarsson, Astrid Gräslund, G. Horváth, B. Libisch, Z. Gombos, N. V. Budagovskaya, N. Kudryasheva, Erisa Harada, Yuki Fukuoka, Tomoaki Ohmura, Arima Fukunishi, Gota Kawai, Kimitsuna Watanabe, Jure Derganc, Bojan Božič, Saša Svetina, Boštjan Žekš, J. F. Y. Hoh, Z. B. Li, G. H. Rossmanith, E. L. de Beer, B. W. Treijtel, P. L. T. M. Frederix, T. Blangè, S. Hénon, F. Galtet, V. Laurent, E. Planus, D. Isabey, L. S. Rath, P. K. Dash, M. K. Raval, C. Ramakrishnan, R. Balaram, Milan Randic, Subhash C. Basak, Marjan Vracko, Ashesh Nandy, Dragan Amic, Drago Beslo, Sonja Nikolic, Nenad Trinajstic, J. Walahaw, Marc F. J. Lensink, Boojala V. B. Reddy, Ilya N. Shindylov, Philip E. Bourne, M. C. Donnamaria, J. de Xammar Oro, J. R. Grigera, Monica Neagu, Adrian Neagu, Matej Praprotnik, Dušanka Janežič, Pekka Mark, Lennart Nilsson, L. La Fata, Laurent E. Dardenne, Araken S. Werneck, Marçal de O. Neto, N. Kannan, S. Vishveshwara, K. Veluraja, Gregory D. Grunwald, Alexandra T. Balaban, Kanika Basak, Brian D. Gute, Denise Mills, David Opitz, Krishnan Balasubramanian, G. I. Mihalas, Diana Lungeanu, G. Macovievici, Raluca Gruia, C. Cortez-Maghelly, B. Dalcin, E. P. Passos, S. Blesic, M. Ljubisavljevic, S. Milosevic, D. J. Stratimirovic, Nandita Bachhawat, Shekhar C. Mande, A. Nandy, Ayumu Saito, Koichi Nishigaki, Mohammed Naimuddin, Takatsugu Hirokawa, Mitsuo Ono, Hirotomo Takaesu, M. I. El Gohary, Abdalla S. Ahmed, A. M. Eissa, Hiroshi Nakashima, G. P. S. Raghava, N. Kurgalvuk, O. Goryn, Bernard S. Gerstman, E. V. Gritsenko, N. N. Remmel, O. M. Maznyak, V. A. Kratasyuk, E. N. Esimbekova, D. Tchitchkan, S. Koulchitsky, A. Tikhonov, A. German, Y. Pesotskaya, S. Pashkevich, S. Pletnev, V. Kulchitsky, Umamaheswar Duvvuri, Sridhar Charagundla, Rahim Rizi, John S. Leigh, Ravinder Reddy, Mahesh Kumar, O. Coshic, P. K. Julka, O. K. Rath, NR. Jagannathan, Karina Roxana Iliescu, Maria Sajin, Nicolcta Moisoi, Ileana Petcu, A. I. Kuzmenko, R. P. Morozova, I. A. Nikolenko, G. V. Donchenko, M. K. Rahman, M. M. Ahmed, Takehiro Watanabe, Y. Rubin, H. Gilboa, R. Sharony, R. Ammar, G. Uretzky, M. Khubchandani, H. N. Mallick, V. Mohan Kumar, Arijitt Borthakur, Erik M. Shapiro, M. Gulnaz Begum, Mahaveer N. Degaonkar, S. Govindasamy, Ivan Dimitrov, T. A. Kumosani, W. Bild, I. Stefanescu, G. Titescu, R. Iliescu, C. Lupusoru, V. Nastasa, I. Haulica, Gopal Khetawat, N. Faraday, M. Nealen, S. Noga, P. F. Bray, T. V. Ananieva, E. A. Lycholat, MV. Kosevich, S. G. Stepanyan, S. V. Antonyuk, R. Khachatryan, H. Arakelian, A. Kumar, S. Ayrapetyan, V. Mkheyan, S. Agadjanyan, A. Khachatryan, S. S. Rajan, V. Kabaleeswaran, Geetha Gopalakrishnan, T. R. Govindachari, Meera Ramrakhiani, Phillip Lowe, Andrew Badley, David C. Cullen, H. Hermel, W. Schmahl, H. Möhwald, Nirmalya Majumdar, Joydip Das, András Dér, Loránd Kelemen, László Oroszi, András Hámori, Jeremy J. Ramsden, Pál Ormos, D. Savitri, Chanchal K. Mitra, Toshio Yanagida, Seiji Esaki, Yuji Kimura, Tomoyuki Nishida, Yosiyuki Sowa, M. Radu, V. K. Koltover, Ya. I. Estrin, L. A. Kasumova, V. P. Bubnov, E. E. Laukhina, Rajiv Dotta, M. Degaonkar, P. Raghunathan, Rama Jayasundar, Pavel Novák, Milan Marko, Ivan Zahradník, Hiroaki Hirata, Hidetake Miyata, J. Balaji, P. Sengupta, S. Maiti, M. Gonsalves, A. L. Barker, J. V. Macpherson, D. O’Hare, C. P. Winlove, P. R. Unwin, R. Phillip, S. Banerjee, G. Ravindra Kumar, K. Nagayaka, R. Danev, S. Sugitani, K. Murata, Michael Gősch, H. Blom, P. Thyberg, Z. Földes-Papp, G. Björk, J. Holm, T. Heino, Masashi Yokochi, Fuyuhiko Inagaki, Masami Kusunoki, E. K. Matthews, J. Pines, Yu. P. Chukova, Vitaly K. Koltover, Geetanjali Bansal, Uma Singh, M. P. Bansal, Kotoko Nakata, Tastuya Nakano, Tsuguchika Kaminuma, B. P. S. Kang, U. Singh, Bonn Kirn, Neja Potocnik, Vito Stare, Latal Shukla, V. Natarajan, T. P. A. Devasagayam, M. D. Sastry, P. C. Kesavan, R. Sayfutdinov, V. V. Adamovich, D. Yu. Rogozin, A. G. Degermendzhy, C. L. Khetrapal, G. A. Nagana Gowda, Kedar Nath Ghimire, Ishida Masaru, H. Fujita, S. Ishiwata, Y. Kishimoto, S. Kawahara, M. Suzuki, H. Mori, M. Mishina, Y. Kirino, H. Ohshima, A. S. Dukhin, V. N. Shilov, P. J. Goetz, and R. K. Mishra

Subjects
0303 health sciences, biology, General Medicine, 010402 general chemistry, 01 natural sciences, Horseradish peroxidase, General Biochemistry, Genetics and Molecular Biology, 0104 chemical sciences, 03 medical and health sciences, Biochemistry, Manganese porphyrin, biology.protein, Enzyme reconstitution, General Agricultural and Biological Sciences, 030304 developmental biology
Published
1999
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15. A Simplified Method for the Efficient Refolding and Purification of Recombinant Human GM-CSF

Author

Melanie Olson, John W. Schrader, Christy A. Thomson, and Linda M. Jackson

Subjects
Protein Folding, lcsh:Medicine, medicine.disease_cause, Protein Engineering, Biochemistry, Inclusion bodies, Mass Spectrometry, law.invention, 0302 clinical medicine, law, Cloning, Molecular, lcsh:Science, Inclusion Bodies, 0303 health sciences, Multidisciplinary, Immune System Proteins, Hematology, Recombinant Proteins, Haematopoiesis, Granulocyte macrophage colony-stimulating factor, 030220 oncology & carcinogenesis, Recombinant DNA, Cytokines, Medicine, Protein folding, medicine.drug, Research Article, Proteases, Immunology, Biophysics, Rheumatoid Arthritis, Biology, 03 medical and health sciences, Rheumatology, Growth Factors, medicine, Escherichia coli, Humans, 030304 developmental biology, DNA Primers, lcsh:R, Proteins, Granulocyte-Macrophage Colony-Stimulating Factor, Protein engineering, Hematopoiesis, Immune System, lcsh:Q
Abstract

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.

Published
2012

16. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

Author

Emil F. Pai, Christy A. Thomson, Steve Bryson, John W. Schrader, A. Louise Creagh, and Gary R. McLean

Subjects
Human cytomegalovirus, Models, Molecular, Molecular Sequence Data, Immunoglobulin Variable Region, Cytomegalovirus, General Biochemistry, Genetics and Molecular Biology, Germline, Epitope, Article, Antigen, medicine, Humans, Amino Acid Sequence, Binding site, Neutralizing antibody, Molecular Biology, Gene, Genetics, General Immunology and Microbiology, biology, Genes, Immunoglobulin, General Neuroscience, Antibodies, Monoclonal, medicine.disease, Virology, Protein Structure, Tertiary, biology.protein, Thermodynamics, Binding Sites, Antibody, Antibody
Abstract

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

Published
2008

17. Identification of small molecule chemical inhibitors of the collagen-specific chaperone Hsp47

Author

Christy A, Thomson, Helen M, Atkinson, and Vettai S, Ananthanarayanan

Subjects
Pyridazines, Thiazoles, Databases, Factual, Nephelometry and Turbidimetry, Collagen, Thiophenes, Sulfides, Heat-Shock Proteins, Serpins, Molecular Chaperones
Abstract

Hsp47 is a collagen-specific molecular chaperone whose activity has been implicated in the pathogenesis of fibrotic diseases. Here, we describe the development of an assay for screening libraries of chemical compounds for inhibitors of Hsp47. A preliminary screen of 2080 compounds identified four that demonstrated inhibitory activity against Hsp47 in vitro, with IC(50) values ranging from 3 to 27 muM. Compounds identified through this method may provide the basis for development of novel antifibrotic therapeutics.

Published
2005

18. Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

Author

Vettai S. Ananthanarayanan, Christy A. Thomson, and Ruggero Tenni

Subjects
animal structures, Type II collagen, Peptide, Plasma protein binding, Biochemistry, Binding, Competitive, Peptide Mapping, Collagen Type I, Article, Substrate Specificity, chemistry.chemical_compound, Radioligand Assay, Sulfur Isotopes, medicine, Collagenases, Cyanogen Bromide, Binding site, Molecular Biology, Collagen Type II, Heat-Shock Proteins, chemistry.chemical_classification, Circular Dichroism, Peptide Fragments, Procollagen peptidase, chemistry, embryonic structures, Collagenase, Cyanogen bromide, Type I collagen, medicine.drug, Molecular Chaperones, Protein Binding
Abstract

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.

Published
2003

19. A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone

Author

Vettai S. Ananthanarayanan and Christy A. Thomson

Subjects
animal structures, Lysis, Trimer, Biology, medicine.disease_cause, Sulfur Radioisotopes, Collagen Type I, Adduct, Affinity chromatography, medicine, Escherichia coli, Animals, Cloning, Molecular, HSP47 Heat-Shock Proteins, Heat-Shock Proteins, Temperature, Rats, Electrophoresis, Biochemistry, Chaperone (protein), embryonic structures, biology.protein, Intein, Biotechnology, Molecular Chaperones, Protein Binding
Abstract

Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions. We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain. Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol. Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies. Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used. The ability to form a trimer may be important for its role as a chaperone. The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments. Illustrative data on collagen binding by (35)S-Hsp47 are shown.

Published
2001

20. Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro

Author

Christy A. Thomson and Vettai S. Ananthanarayanan

Subjects
Conformational change, animal structures, Protein Conformation, Fibril, Biochemistry, symbols.namesake, Structure-Activity Relationship, Heat shock protein, Animals, Molecular Biology, HSP47 Heat-Shock Proteins, Heat-Shock Proteins, Chemistry, Endoplasmic reticulum, Circular Dichroism, Cell Biology, Golgi apparatus, Hydrogen-Ion Concentration, In vitro, Recombinant Proteins, Rats, Procollagen peptidase, Spectrometry, Fluorescence, embryonic structures, symbols, Collagen, Type I collagen, Research Article
Abstract

Hsp47, a 47kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6–7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10min and levels off by approx. 60min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2–7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in α-helix and an increase in β-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions.

Published
2000

21. CS05-2. Genetic and functional analysis of a panel of truly human monoclonal antibodies against GM-CSF generated from idiopathic pulmonary alveolar proteinosis (IPAP) patients and potential therapeutic uses as an anti-inflammatory agent

Author

John W. Schrader, John Seymour, Gary P. Anderson, Lenka Allan, John A. Hamilton, Yanni Wang, Linda M. Jackson, Amanda L Turner, Thomas K. Waddell, and Christy A. Thomson

Subjects
Pathology, medicine.medical_specialty, medicine.drug_class, business.industry, Immunology, Hematology, Monoclonal antibody, medicine.disease, Biochemistry, Anti-inflammatory, medicine, Immunology and Allergy, Pulmonary alveolar proteinosis, business, Molecular Biology
Published
2011
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