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Gunnarsson, Lavinia
Publications (9 of 9) Show all publications
Hagemann, U. B., Gunnarsson, L., Geraudie, S., Scheffler, U., Griep, R. A., Reiersen, H., . . . Kiprijanov, S. M. (2014). Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis. PLoS ONE, 9(7), Article ID e103776.
Open this publication in new window or tab >>Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, article id e103776Article in journal (Refereed) Published
Abstract [en]

Background: CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis.

Methodology: Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.

Significance: For the first time, successful generation of anti-G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library Science, 2014
National Category
Medical and Health Sciences Immunology Cell Biology
Research subject
Immunology
Identifiers
urn:nbn:se:oru:diva-45516 (URN)10.1371/journal.pone.0103776 (DOI)000339954800080 ()25080123 (PubMedID)2-s2.0-84905119945 (Scopus ID)
Available from: 2015-08-11 Created: 2015-08-11 Last updated: 2017-12-04Bibliographically approved
von Schantz, L., Gullfot, F., Scheer, S., Filonova, L., Gunnarsson, L. C., Flint, J. E., . . . Ohlin, M. (2009). Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules. BMC Biotechnology, 9, Article ID 92.
Open this publication in new window or tab >>Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules
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2009 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 9, article id 92Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.

RESULTS: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections.

CONCLUSION: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

Place, publisher, year, edition, pages
BioMed Central, 2009
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64486 (URN)10.1186/1472-6750-9-92 (DOI)000272337000001 ()19878581 (PubMedID)2-s2.0-71049184894 (Scopus ID)
Funder
Swedish Research CouncilMarianne and Marcus Wallenberg Foundation
Note

Funding Agencies:

Formas Funcfiber Centre

NSF  RCN-0090281

Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-26Bibliographically approved
Gunnarsson, L. C., Montanier, C., Tunnicliffe, R. B., Williamson, M. P., Gilbert, H. J., Nordberg Karlsson, E. & Ohlin, M. (2007). Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module. Biochemical Journal, 406(2), 209-214
Open this publication in new window or tab >>Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module
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2007 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 406, no 2, p. 209-214Article in journal (Refereed) Published
Abstract [en]

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

Place, publisher, year, edition, pages
Portland Press, 2007
Keywords
aromatic residue; binding specificity; carbohydrate-binding module; molecular engineering; thermodynamics; xylan
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64488 (URN)10.1042/BJ20070128 (DOI)000249181200003 ()17506724 (PubMedID)2-s2.0-34548178992 (Scopus ID)
Note

Funding Agency:

Biotechnology and Biological Sciences Research Council  BB/C005074/1

Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-26Bibliographically approved
Filonova, L., Gunnarsson, L. C., Daniel, G. & Ohlin, M. (2007). Synthetic xylan-binding modules for mapping of pulp fibres and wood sections. BMC Plant Biology, 7, Article ID 54.
Open this publication in new window or tab >>Synthetic xylan-binding modules for mapping of pulp fibres and wood sections
2007 (English)In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 7, article id 54Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.

RESULTS: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

CONCLUSION: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

Place, publisher, year, edition, pages
BioMed Central, 2007
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64435 (URN)10.1186/1471-2229-7-54 (DOI)000251529800001 ()17935619 (PubMedID)2-s2.0-36749005056 (Scopus ID)
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-24Bibliographically approved
Gunnarsson, L. C., Zhou, Q., Montanier, C., Karlsson, E. N., Brumer, H. & Ohlin, M. (2006). Engineered xyloglucan specificity in a carbohydrate-binding module. Glycobiology, 16(12), 1171-1180
Open this publication in new window or tab >>Engineered xyloglucan specificity in a carbohydrate-binding module
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2006 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 16, no 12, p. 1171-1180Article in journal (Refereed) Published
Abstract [en]

The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

Place, publisher, year, edition, pages
Oxford University Press, 2006
Keywords
binding specificity; carbohydrate-binding module; molecular engineering; phage display; xyloglucan
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64482 (URN)10.1093/glycob/cwl038 (DOI)000242270800002 ()16902199 (PubMedID)2-s2.0-33751368319 (Scopus ID)
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-26Bibliographically approved
Gunnarsson, L. C., Dexlin, L., Karlsson, E. N., Holst, O. & Ohlin, M. (2006). Evolution of a carbohydrate binding module into a protein-specific binder. Biomolecular Engineering, 23(2-3), 111-117
Open this publication in new window or tab >>Evolution of a carbohydrate binding module into a protein-specific binder
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2006 (English)In: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 23, no 2-3, p. 111-117Article in journal (Refereed) Published
Abstract [en]

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.

Place, publisher, year, edition, pages
Elsevier, 2006
Keywords
binding specificity; combinatorial library; mutant; phage-display; molecular engineering; protein scaffold
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64485 (URN)10.1016/j.bioeng.2005.12.002 (DOI)000237050100004 ()16427804 (PubMedID)2-s2.0-33645457940 (Scopus ID)
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-25Bibliographically approved
Gunnarsson, L. C., Nordberg Karlsson, E., Andersson, M., Holst, O. & Ohlin, M. (2006). Molecular engineering of a thermostable carbohydrate-binding module. Paper presented at 6th Carbohydrate Bioengineering Meeting (CBM6), Barcelona, Spain, April 3-6, 2005. Biocatalysis and Biotransformation, 24(1-2), 31-37
Open this publication in new window or tab >>Molecular engineering of a thermostable carbohydrate-binding module
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2006 (English)In: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 24, no 1-2, p. 31-37Article in journal (Refereed) Published
Abstract [en]

Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities.

Place, publisher, year, edition, pages
Informa Healthcare, 2006
Keywords
binding specificity; carbohydrate-binding module; combinatorial library; molecular engineering; phage-display; protein scaffold
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64490 (URN)10.1080/10242420500518516 (DOI)000239089700005 ()2-s2.0-33646838036 (Scopus ID)
Conference
6th Carbohydrate Bioengineering Meeting (CBM6), Barcelona, Spain, April 3-6, 2005
Funder
Swedish Research Council
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-02-20Bibliographically approved
Johansson, R., Gunnarsson, L. C., Ohlin, M. & Ohlson, S. (2006). Thermostable carbohydrate-binding modules in affinity chromatography. Journal of Molecular Recognition, 19(4), 275-281
Open this publication in new window or tab >>Thermostable carbohydrate-binding modules in affinity chromatography
2006 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 19, no 4, p. 275-281Article in journal (Refereed) Published
Abstract [en]

Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate-binding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based oligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C.

With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-microM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2006
Keywords
affinity chromatography; carbohydrate-binding modules; thermostable; weak affinity
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64484 (URN)10.1002/jmr.794 (DOI)000239882300005 ()16838297 (PubMedID)2-s2.0-33747274375 (Scopus ID)
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-26Bibliographically approved
Gunnarsson, L. C., Nordberg Karlsson, E., Albrekt, A.-S., Andersson, M., Holst, O. & Ohlin, M. (2004). A carbohydrate binding module as a diversity-carrying scaffold. Protein Engineering Design & Selection, 17(3), 213-221
Open this publication in new window or tab >>A carbohydrate binding module as a diversity-carrying scaffold
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2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 3, p. 213-221Article in journal (Refereed) Published
Abstract [en]

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

Place, publisher, year, edition, pages
Oxford University Press, 2004
Keywords
binding specificity; carbohydrate binding module; molecular library; phage selection; scaffold
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-64487 (URN)10.1093/protein/gzh026 (DOI)000222797300003 ()15082834 (PubMedID)2-s2.0-2942748131 (Scopus ID)
Available from: 2018-01-24 Created: 2018-01-24 Last updated: 2018-01-26Bibliographically approved
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