| 1. |
- Ellmark, Peter, et al.
(författare)
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A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement
- 2004
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 17:4, s. 316-322
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Tidskriftsartikel (refereegranskat)abstract
- The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component. Copyright (C) 2004 John Wiley Sons, Ltd.
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| 2. |
- Ellmark, Peter, et al.
(författare)
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In vitro molecular evolution of antibody genes mimicking receptor revision
- 2002
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Ingår i: Molecular Immunology. - 1872-9142. ; 39:5-6, s. 349-356
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Tidskriftsartikel (refereegranskat)abstract
- Antibody evolution in vivo proceeds mainly by stepwise improvements, accomplished by single base pair substitutions. Lately, receptor revision, i.e. exchange of large parts of the V gene for another sequence, has been suggested to provide a complementary route for affinity maturation. By employing a receptor revision like evolution process in vitro using combinatorial libraries and phage display selection, we demonstrate here that maturation of a clone may preferentially proceed through exchange of a large gene segment rather than via minor sequence changes. These modifications of a CD40-specific human antibody fragment outline how receptor revision like events may provide an advantage to a particular clonotype put under selective pressure. (C) 2002 Elsevier Science Ltd. All rights reserved.
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| 3. |
- Ellmark, Peter, et al.
(författare)
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Modulation of the CD40-CD40 ligand interaction using human anti-CD40 single-chain antibody fragments obtained from the n-CoDeR phage display library
- 2002
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Ingår i: Immunology. - 0019-2805. ; 106:4, s. 456-463
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Tidskriftsartikel (refereegranskat)abstract
- CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.
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| 4. |
- Ellmark, Peter, et al.
(författare)
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Pre-assembly of the extracellular domains of CD40 is not necessary for rescue of mouse B cells from anti-immunoglobulin M-induced apoptosis
- 2003
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Ingår i: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 108:4, s. 452-457
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Tidskriftsartikel (refereegranskat)abstract
- CD40 is a tumour necrosis factor receptor (TNFR) family member of central importance for the adaptive immune system. To elucidate the functional role of the different extracellular domains of CD40, we have created a set of truncated CD40 molecules where domains, or parts of domains, have been removed. These CD40 proteins, which contain a peptide tag in the N-terminal end, have been expressed in a murine B-cell line, WEHI 231. It was found that ligation of these engineered CD40 proteins via the peptide tag, was sufficient to rescue as well as to promote proliferation of apoptotic WEHI 231 cells, even when all the extracellular domains of CD40 were absent. Our results suggest that pre association of CD40 in the cell membrane plays no critical role for the CD40 signalling pathway. Furthermore, our data imply that conformational changes initiated in the extracellular domains of CD40 are not essential for signal transduction.
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| 5. |
- Fransson, Johan, et al.
(författare)
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Internalising human anti-tumor antibodies.
- 2003
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Ingår i: Immunology Letters (Abstracts of the 15th European Immunology Congress (EFIS 2003)). - 1879-0542 .- 0165-2478. ; 87:1-3, s. 02-34
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 6. |
- 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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| 7. |
- Furebring, Christina, et al.
(författare)
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Antibody-mediated neutralization of cytomegalovirus: modulation of efficacy induced through the IgG constant region.
- 2002
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Ingår i: Molecular Immunology. - 1872-9142. ; 38:11, s. 833-840
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies can neutralize the infectious properties of human cytomegalovirus (CMV). In vivo, the major neutralization determinants are located on glycoprotein B (gB). Recombinant human antibodies, that carry different constant regions (IgG1, IgG3 and the synthetic variant IgG3mA) against two of these epitopes were investigated for their ability to recruit the complement cascade for destruction of the virus. It was shown that all variants of an antibody against the antigenic domain (AD)-2 epitope displayed a similar neutralization activity despite the fact that improved C1q binding was observed for IgG3 and IgG3mA over the IgG1 variant. In contrast, an antibody against the AD-1 epitope carrying the normal IgG3 constant region, was less efficient than its IgG1 counterpart in neutralizing the virus in the absence of complement. However, it restored its activity in the presence of complement to the level of the naturally occurring IgG1 version. The same antibody was substantially more potent in neutralizing the virus in the presence of complement if it carried the IgG3mA constant region. This demonstrates the importance of the constant domain for the biological activity of AD-1 specific antibodies, a factor that should be taken into account when using antibody-based therapeutics or when inducing antibodies by vaccination.
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| 8. |
- Malmborg Hager, Ann-Christin, et al.
(författare)
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Affinity and Epitope Profiling of Mouse Anti-CD40 Monoclonal Antibodies
- 2003
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 1365-3083 .- 0300-9475. ; 57:6, s. 517-524
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Tidskriftsartikel (refereegranskat)abstract
- The CD40-CD40L interaction plays a critical role in both humoral and cellular immune responses and interfering antibodies have been suggested as an effective approach for the treatment of lymphomas and autoimmune diseases. In this study we have profiled a panel of mouse antihuman CD40 monoclonal antibodies (MoAbs), regarding their CD40 binding affinity and epitope-specificity relative to the CD40L binding in relation to their cellular activating potential. Despite a rather similar domain-recognition profile, the MoAbs blocked the CD40L binding to a varying degree, with MoAb 5C3 being the poorest inhibitor. There was no correlation between affinity and cellular activation potential. In contrast, a correlation between the ability to block CD40L-binding and activation potential could be seen. We believe that this analysis of several mouse anti-CD40 antibodies can be used to develop strategies for producing new human anti-CD40 antibodies that can more effectively induce or block B-cell proliferation.
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