| 1. |
- Kristan, Matej, et al.
(författare)
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The first visual object tracking segmentation VOTS2023 challenge results
- 2023
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Ingår i: 2023 IEEE/CVF International conference on computer vision workshops (ICCVW). - : Institute of Electrical and Electronics Engineers Inc.. - 9798350307443 - 9798350307450 ; , s. 1788-1810
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Konferensbidrag (refereegranskat)abstract
- The Visual Object Tracking Segmentation VOTS2023 challenge is the eleventh annual tracker benchmarking activity of the VOT initiative. This challenge is the first to merge short-term and long-term as well as single-target and multiple-target tracking with segmentation masks as the only target location specification. A new dataset was created; the ground truth has been withheld to prevent overfitting. New performance measures and evaluation protocols have been created along with a new toolkit and an evaluation server. Results of the presented 47 trackers indicate that modern tracking frameworks are well-suited to deal with convergence of short-term and long-term tracking and that multiple and single target tracking can be considered a single problem. A leaderboard, with participating trackers details, the source code, the datasets, and the evaluation kit are publicly available at the challenge website1
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| 2. |
- Yu, Wendy, et al.
(författare)
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Efficient Labeling of Native Human IgG by Proximity-Based Sortase-Mediated Isopeptide Ligation
- 2021
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 32:6, s. 1058-1066
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-drug conjugates (ADCs) have demonstrated great therapeutic potential due to their ability to target the delivery of potent cytotoxins. However, the heterogeneous nature of conventional drug conjugation strategies can affect the safety, efficacy, and stability of ADCs. Site-specific conjugations can resolve these issues, but often require genetic modification of Immunoglobulin G (IgG), which can impact yield or cost of production, or require undesirable chemical linkages. Here, we describe a near-traceless conjugation method that enables the efficient modification of native IgG, without the need for genetic engineering or glycan modification. This method utilizes engineered variants of sortase A to catalyze noncanonical isopeptide ligation. Sortase A was fused to an antibody-binding domain to improve ligation efficiency. Antibody labeling is limited to five lysine residues on the heavy chain and one on the light chain of human IgG1. The ADCs exhibit conserved antigen and Fc-receptor interactions, as well as potent cytolytic activity.
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| 3. |
- Grimm, Sebastian, et al.
(författare)
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Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
- 2011
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Ingår i: Molecular Biotechnology. - : Springer Science and Business Media LLC. - 1073-6085 .- 1559-0305. ; 48:3, s. 263-276
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Tidskriftsartikel (refereegranskat)abstract
- Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.
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| 4. |
- Grimm, Sebastian, et al.
(författare)
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Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784. ; 27:6, s. 766-773
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Tidskriftsartikel (refereegranskat)abstract
- Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed
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| 5. |
- Tao, Liang, et al.
(författare)
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Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits similar to 11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.
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| 6. |
- Yu, Feifan, et al.
(författare)
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An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
- 2014
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Ingår i: mAbs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 6:6, s. 1598-1607
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Tidskriftsartikel (refereegranskat)abstract
- In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor a (sIL-6R alpha) and IL6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z(IL-6_13) with an affinity (K-D) for IL-6 of similar to 500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, Z(IL-6_13) was fused to either the N-or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira (R)). One AffiMab construct with Z(IL-6_13) positioned at the N-terminus of the heavy chain, denoted Z(IL-6_13)-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z(IL-6_13)-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.
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| 7. |
- Yu, Feifan, 1983-
(författare)
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Generating Affinity Proteins for Biotechnological, Diagnostic and Therapeutic Applications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein engineering is a powerful tool to modify proteins to generate novel and desired properties that could be applied in biotechnological, diagnostics and therapeutic areas. In this thesis, both rational design and library based engineering principles have been exploited to develop affinity proteins with desired traits.One study was focused on the use of site-directed mutagenesis to obtain variants of the staphylococcal protein A-derived 58-residue immunoglobulin binding Z domain with improved affinity for mouse IgG1 Fc. Screening of ca. 170 constructed variants revealed one variant with a single F5I amino acid substitution, denoted ZF5I, with a ten-fold higher affinity. The Fc binding ZF5I variant was further investigated for use in affinity-driven site-specific covalent photoconjugation to mIgG1 monoclonal antibodies. Here, nine candidate positions in the domain were investigated for introduction of a UV-activatable maleimide benzophenone (MBP) group via conjugation to an introduced cysteine residue. The best photo-conjugation results were obtained for a variant in which the MBP was introduced at position 32, denoted ZF5I-Q32C-MBP, which could be conjugated at high yields to all nineteen mouse IgG1s tested. The use of a biotinylated Z-based probe for biotinylation via photoconjugation of a monoclonal anti-interferon gamma antibody resulted in a higher antigen binding activity than if a conventional amine directed biotinylation strategy was used.In a second study, the goal was to develop a new homogeneous immunoassay for quick antigen detection, based on split-protein complementation and pairs of antigen recognizing proteins. In one of the formats investigated, separate fragments of a split-beta-lactamase enzyme reporter were genetically linked to ZF5I-Q32C-MBP units which were individually photo-conjugated to two different mAbs recognizing different epitopes on a human interferon gamma model target analyte. Simultaneous binding of the two mAb-enzyme half probes to the analyte resulted in an analyte concentration-dependent enzyme fragment complementation which could be spectrophotometrically detected using a nitrocefin substrate.Using ribosome display technology, Z-domain based binders to mouse IgG1 were selected from an affibody library. One binder denoted Zmab25 was shown capable of selective binding to mouse IgG in a background of bovine IgG, and could be used for species-selective recovery of monoclonal antibodies from complex samples, resembling hybridoma culture supernatants. Epitope mapping experiments showed that that the binding site on mouse IgG was located in the Fab fragment and was overlapping with that of streptococcal protein G.In a final study, phage display technology was used to select affibodies binding to human interleukin 6 (IL-6), for potential use in rheumatoid arthritis (RA) therapy via blocking of the signaling involving the ternary complex between IL-6, the IL-6 receptor α (IL-6R α) and the gp130 co-receptor. Several affibodies were shown to be capable of blocking the interaction between gp130 and preformed complexes of IL-6 and soluble IL-6R α (IL-6/sIL-6R α) in vitro, corresponding to the so-called trans-signaling interaction. One of these affibody variants denoted ZIL-6_13 showed a KD of approx. 500 pM for IL-6 and was genetically fused to different chain ends of the monoclonal anti-TNF antibody adalimumab to build bi-specific “AffiMab” constructs. One construct, ZIL-6_13-HCAda,in which the affibody was fused to the N-terminus of the adalimumab heavy chain had the most optimal properties in different cell assays and was also evaluated in vivo in an acute serum amyloid A (SAA) mouse RA model, involving a dual challenge of animals with both IL-6 and TNF. Compared to adalimumab that could only reduce SAA levels to 50% at the highest dose, the bi-functional AffiMab reduced SAA levels to below the detection level.
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| 8. |
- Yu, Feifan, et al.
(författare)
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Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation
- 2018
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Ingår i: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 13:7
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Tidskriftsartikel (refereegranskat)abstract
- Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.
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| 9. |
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| 10. |
- Yu, Feifan, et al.
(författare)
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Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:2, s. e56597-
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG(1) (mIgG(1)). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG(1) monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG(1), The best photocoupling efficiency to mIgG(1) Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG(1) mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG(1) antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG(1) mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly h`igher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.
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