| 1. |
- Nilvebrant, Johan, 1982-, et al.
(författare)
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An introduction to epitope mapping
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Humana Press. - 1064-3745 .- 1940-6029. ; 1785, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are protein molecules used routinely for therapeutic, diagnostic, and research purposes due to their exquisite ability to selectively recognize and bind a given antigen. The particular area of the antigen recognized by the antibody is called the epitope, and for proteinaceous antigens the epitope can be of complex nature. Information about the binding epitope of an antibody can provide important mechanistic insights and indicate for what applications an antibody might be useful. Therefore, a variety of epitope mapping techniques have been developed to localize such regions. Although the real picture is even more complex, epitopes in protein antigens are broadly grouped into linear or discontinuous epitopes depending on the positioning of the epitope residues in the antigen sequence and the requirement of structure. Specialized methods for mapping of the two different classes of epitopes, using high-throughput or high-resolution methods, have been developed. While different in their detail, all of the experimental methods rely on assessing the binding of the antibody to the antigen or a set of antigen mimics. Early approaches utilizing sets of truncated proteins, small numbers of synthesized peptides, and structural analyses of antibody-antigen complexes have been significantly refined. Current state-of-the-art methods involve combinations of mutational scanning, protein display, and high-throughput screening in conjunction with bioinformatic analyses of large datasets.
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| 2. |
- Rockberg, Johan, et al.
(författare)
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Preface
- 2018
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Ingår i: Methods in Molecular Biology. - : Humana Press. ; , s. v-vi
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Anfelt, Josefine, et al.
(författare)
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Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.
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| 4. |
- Forsström, Björn, et al.
(författare)
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Dissecting Antibodies with Regards to Linear and Conformational Epitopes
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.
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| 5. |
- Hansen, Henning Gram, et al.
(författare)
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Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
- 2015
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.
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| 6. |
- Hu, Francis Jingxin, 1986-, et al.
(författare)
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Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
- 2018
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 45, s. 80-88
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Tidskriftsartikel (refereegranskat)abstract
- Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.
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| 7. |
- Hu, Francis Jingxin, 1986-, et al.
(författare)
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SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping
- 2019
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 47:6
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Tidskriftsartikel (refereegranskat)abstract
- Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.
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| 8. |
- Hu, Francis Jingxin, 1986-
(författare)
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Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and Characterization
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine.
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| 9. |
- Häussler, Ragna S., et al.
(författare)
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Systematic Development of Sandwich Immunoassays for the Plasma Secretome
- 2019
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861.
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Tidskriftsartikel (refereegranskat)abstract
- The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.
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| 10. |
- Klevebring, Daniel, et al.
(författare)
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Exome sequencing of contralateral breast cancer identifies metastatic disease
- 2015
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Ingår i: Breast Cancer Research and Treatment. - Stockholm : Karolinska Institutet, Dept of Medical Epidemiology and Biostatistics. - 0167-6806 .- 1573-7217.
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Tidskriftsartikel (refereegranskat)abstract
- Women with contralateral breast cancer (CBC) have significantly worse prognosis compared to women with unilateral cancer. A possible explanation of the poor prognosis of patients with CBC is that in a subset of patients, the second cancer is not a new primary tumor but a metastasis of the first cancer that has potentially obtained aggressive characteristics through selection of treatment. Exome and whole-genome sequencing of solid tumors has previously been used to investigate the clonal relationship between primary tumors and metastases in several diseases. In order to assess the relationship between the first and the second cancer, we performed exome sequencing to identify somatic mutations in both first and second cancers, and compared paired normal tissue of 25 patients with metachronous CBC. For three patients, we identified shared somatic mutations indicating a common clonal origin thereby demonstrating that the second tumor is a metastasis of the first cancer, rather than a new primary cancer. Accordingly, these patients all developed distant metastasis within 3 years of the second diagnosis, compared with 7 out of 22 patients with non-shared somatic profiles. Genomic profiling of both tumors help the clinicians distinguish between true CBCs and subsequent metastases.
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| 11. |
- Ko, Bong-Kook, et al.
(författare)
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Combination of novel HER2-targeting antibody 1E11 with trastuzumab shows synergistic antitumor activity in HER2-positive gastric cancer
- 2015
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Ingår i: Molecular Oncology. - : Elsevier. - 1878-0261 .- 1574-7891. ; 9:2, s. 398-408
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Tidskriftsartikel (refereegranskat)abstract
- The synergistic interaction of two antibodies targeting the same protein could be developed as an effective anti-cancer therapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast and gastric cancer patients, and HER2-targeted antibody therapy using trastuzumab is effective in many of these patients. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2-positive gastric cancer. Here, we describe the development of 1E11, a HER2-targeted humanized monoclonal antibody showing increased efficacy in a highly synergistic manner in combination with trastuzumab in the HER2-overexpressing gastric cancer cell lines NCI-N87 and OE-19. The two antibodies bind to sub-domain IV of the receptor, but have non-overlapping epitopes, allowing them to simultaneously bind HER2. Treatment with 1E11 alone induced apoptosis in HER2-positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. The synergistic antitumor activity of 1E11 in combination with trastuzumab indicates that it could be a novel potent therapeutic antibody for the treatment of HER2-overexpressing gastric cancers.
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| 12. |
- Lundqvist, Magnus, et al.
(författare)
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Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.
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| 13. |
- Lundqvist, Magnus
(författare)
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Methods for cell line and protein engineering
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.
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| 14. |
- Lundqvist, Magnus, et al.
(författare)
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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:7
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Tidskriftsartikel (refereegranskat)abstract
- We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
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| 15. |
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| 16. |
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| 17. |
- Sastry, Anand, et al.
(författare)
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Machine learning in computational biology to accelerate high-throughput protein expression
- 2017
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Ingår i: Bioinformatics. - : Oxford University Press. - 1367-4803 .- 1367-4811. ; 33:16, s. 2487-2495
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Tidskriftsartikel (refereegranskat)abstract
- Motivation: The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Results: Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation.
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| 18. |
- Thul, Peter J., et al.
(författare)
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A subcellular map of the human proteome
- 2017
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 356:6340
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.
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| 19. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 20. |
- Uhlén, Mathias, et al.
(författare)
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Tissue-based map of the human proteome
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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| 21. |
- Volk, Anna-Luisa
(författare)
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Cell line and protein engineering tools for production and characterization of biologics
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups.
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| 22. |
- Volk, Anna-Luisa, et al.
(författare)
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Discontinuous epitope mapping of antibodies using bacterial cell surface display of folded domains
- 2018
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Ingår i: Epitope Mapping Protocols. - New York, NY : Humana Press. - 9781493978397 ; , s. 159-183
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Bokkapitel (refereegranskat)abstract
- Knowledge of the exquisite-binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping of discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping of linear epitopes. We here report on the usage of a bacterial surface display-based method for mapping of structural epitopes by display of folded domains on the surface of Gram positive bacteria, followed by domain-targeted mutagenesis and library analysis for the identification of key-residues by flow sorting and sequencing. Identified clones with reduced affinity are validated by single clone FACS and subsequent full-length expression in mammalian cells for validation.
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| 23. |
- Volk, Anna-Luisa, et al.
(författare)
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Epitope mapping of antibodies using bacterial cell surface display of gene fragment libraries
- 2018
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Ingår i: Epitope Mapping Protocols. - New York, NY : Humana Press. ; , s. 141-157
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Bokkapitel (refereegranskat)abstract
- The unique property of specific high affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe an updated protocol for high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric analysis. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment back to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.
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| 24. |
- Volk, Anna-Luisa, et al.
(författare)
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Stratification of responders towards eculizumab using a structural epitope mapping strategy
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.
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| 25. |
- Zamani, Leila, et al.
(författare)
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High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome
- 2018
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 13:10
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Tidskriftsartikel (refereegranskat)abstract
- The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches 200x10(6)cellsmL(-1). The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production.
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