| 1. |
- Carlsson, Roland, et al.
(författare)
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n-CoDeR concept: unique types of antibodies for diagnostic use and therapy.
- 2001
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Ingår i: Expert Review of Molecular Diagnostics. - 1744-8352. ; 1:1, s. 8-102
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Tidskriftsartikel (refereegranskat)abstract
- The n-CoDeR recombinant antibody gene libraries are built on a single master framework, into which diverse in vivo-formed complementarity determining regions (CDRs) are allowed to recombine. These CDRs are sampled from in vivo-processed and proof-read gene sequences, thus ensuring an optimal level of correctly folded and functional molecules. By the modularized assembly process, up to six CDRs can be varied at the same time, providing a possibility for the creation of a hitherto undescribed genetic and functional variation. The n-CoDeR antibody gene libraries can be used to select highly specific, human antibody fragments with specificities to virtually any antigen, including carbohydrates and human self-proteins and with affinities down into the subnanomolar range. Furthermore, combining CDRs sampled from in vivo-processed sequences into a single framework result in molecules exhibiting a lower immunogenicity compared to normal human immunoglobulins, as determined by computer analyses. The distinguished features of the n-CoDeR libraries in the therapeutic and diagnostic areas are discussed.
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| 2. |
- Fransson, Johan, et al.
(författare)
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Profiling of internalizing tumor-associated antigens on breast and pancreatic cancer cells by reversed genomics
- 2004
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Ingår i: Cancer Letters. - : Elsevier BV. - 1872-7980 .- 0304-3835. ; 208:2, s. 235-242
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Tidskriftsartikel (refereegranskat)abstract
- Human antibodies directed towards functionally associated tumor antigens have great potentials as adjuvant treatment in cancer therapy. Here we describe an efficient subtractive approach to select single chain Fv (scFv) antibodies, specifically binding to unknown rapidly internalizing tumor-associated antigens (TAA) on human breast and pancreatic carcinoma cell lines. After re-engineering the scFv into intact IgG molecules, these fully human antibodies displayed individual binding profiles to TAAs on breast, pancreatic, colorectal and prostate carcinomas, while showing no reactivity to lymphomas. The ability of the selected antibodies to undergo receptor-mediated internalization was verified by confocal microscopy, thus proving our strategy to provide a unique set of human antibodies, potentially useful in immunotherapy. (C) 2003 Elsevier Ltd. All rights reserved.
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| 3. |
- Jirholt, P., et al.
(författare)
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A central core structure in an antibody variable domain determines antigen specificity
- 2001
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 1460-213X. ; 14:1, s. 67-74
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Tidskriftsartikel (refereegranskat)abstract
- Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.
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| 4. |
- Kobayashi, N, et al.
(författare)
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Two-step in vitro antibody affinity maturation enables estradiol-17β assays with more than 10-fold higher sensitivity.
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:3, s. 1027-1038
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Tidskriftsartikel (refereegranskat)abstract
- Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a "wild-type" scFv (scFv#E4-4) composed of V(H) and V(L) domains from a mouse anti-E(2) antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by "CDR-shuffling". Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by "shuffling" into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(L29)Val] that had 5-fold higher affinity (K(a) = 2.6 x 10(8) M(-1)) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V(H) and V(L) of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K(a) = 6.3 x 10(8) M(-1); Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E(2) radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E(2)-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.
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| 5. |
- Malmborg, Ann-Christin, et al.
(författare)
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Selection of binders from phage displayed antibody libraries using the BIAcore(TM) biosensor
- 1996
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 198:1, s. 51-57
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Tidskriftsartikel (refereegranskat)abstract
- In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore(TM) biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore(TM) when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.
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| 6. |
- Moberg, Christina, et al.
(författare)
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De unga gör helt rätt när de stämmer staten
- 2022
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Ingår i: Aftonbladet. - 1103-9000.
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- 1 620 forskare och lärare i forskarvärlden: Vi ställer oss bakom Auroras klimatkrav
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| 7. |
- Söderlind, Eskil, et al.
(författare)
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Complementarity-determining region (CDR) implantation : A theme of recombination
- 1999
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Ingår i: Immunotechnology. - 1380-2933. ; 4:3-4, s. 279-285
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Tidskriftsartikel (refereegranskat)abstract
- A novel technology in the area of antibody engineering has been developed which allows for the creation of new types of antibody molecules. It is called complementarity-determining region (CDR) implantation and permits the random combination of CDR sequences formed in vivo into a single master framework. Thus, totally new gene combinations can be produced and used in selection processes. The result is a genetic variability which is extremely large, even exceeding the natural variability found in the immune system. In this commentary, CDR implantation is presented and the technology is discussed.
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| 8. |
- Söderlind, Eskil, et al.
(författare)
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Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries
- 2000
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 18:8, s. 852-856
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Tidskriftsartikel (refereegranskat)abstract
- We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and funcfional variation in antibody molecules.
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| 9. |
- Söderlind, Eskil, et al.
(författare)
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The immune diversity in a test tube - Non-immunised antibody libraries and functional variability in defined protein scaffolds
- 2001
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Ingår i: Combinatorial Chemistry & High Throughput Screening. - 1386-2073. ; 4:5, s. 409-416
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Tidskriftsartikel (refereegranskat)abstract
- Technologies to develop and evolve the function of proteins and, in particular, antibodies have developed rapidly since the introduction of phage display. Importantly, it has become possible to identify molecules with binding properties that cannot be found by other means. A range of different approaches to create general libraries that are useful for the selection of such molecules specific for essentially any kind of target have emerged. We herein review some of the most prominent approaches in the field and in particular discuss specific features related to the development of antibody libraries based on single antibody framework scaffolds. This approach not only permits identification of a range of specific binders, but also facilitates further evolution of initially derived molecules into molecules with optimised functions.
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