| 21. |
- Giang, Kim Anh, et al.
(författare)
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Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells
- 2023
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 77, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
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| 22. |
- Grimm, Sebastian, et al.
(författare)
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Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 534-542
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Tidskriftsartikel (refereegranskat)abstract
- The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.
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| 23. |
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| 24. |
- Gu, Gucci Jijuan, et al.
(författare)
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Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation
- 2013
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 30:2, s. 144-152
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Tidskriftsartikel (refereegranskat)abstract
- While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.
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| 25. |
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| 26. |
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| 27. |
- Gustavsson, Elin, et al.
(författare)
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Surrogate antigens as targets for proteome-wide binder selection
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 28:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.
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| 28. |
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| 29. |
- Heimersson, Sara, 1984, et al.
(författare)
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Methodological issues in life cycle assessment of mixed-culture polyhydroxyalkanoate production utilising waste as feedstock
- 2014
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 31:4, s. 383-393
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Tidskriftsartikel (refereegranskat)abstract
- Assessing the environmental performance of emerging technologies using life cycle assessment (LCA) can be challenging due to a lack of data in relation to technologies, application areas or other life cycle considerations, or a lack of LCA methodology that address the specific concerns. Nevertheless, LCA can be a valuable tool in the environmental optimisation in the technology development phase. One emerging technology is the mixed-culture production of polyhydroxyalkanoates (PHAs). PHA production by pure microbial cultures has been developed and assessed in several LCAs during the previous decade. Recent developments within mixed-culture PHA production call for environmental assessment to guide in technology development. Mixed-culture PHA production can use the organic content in wastewater as a feedstock; the production may then be integrated with wastewater treatment (WWT) processes. This means that mixed-culture PHA is produced as a by-product from services in the WWT.This article explores different methodological challenges for LCA of mixed-culture PHA production using organic material in wastewater as feedstock.LCAs of both pure- and mixed-culture PHA production were reviewed. Challenges, similarities and differences when assessing PHA production by mixed- or pure-cultures were identified and the resulting implications for methodological choices in LCA were evaluated and illustrated, using a case study with mixed- and pure-culture PHA model production systems, based on literature data.Environmental impacts of processes producing multiple products or services need to be allocated between the different products or services. Such situations occur both in feedstock production and when the studied system is providing multiple functions. The selection of allocation method is shown to determine the LCA results. The type of data used, for electricity in the energy system, is shown to be important for the results, which indicates, a strong regional dependency of results for systems with electricity use as an environmental hot spot. The importance of assessing water use, an environmental impact not assessed by any of the reviewed studies, is highlighted. © 2013 Elsevier B.V.
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| 30. |
- 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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