| 1. |
- Borrebaeck, Carl A K, et al.
(författare)
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Kinetic analysis of recombinant antibody-antigen interactions : Relation between structural domains and antigen binding
- 1992
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Ingår i: Bio/Technology. - : Springer Science and Business Media LLC. - 0733-222X. ; 10:6, s. 697-698
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Tidskriftsartikel (refereegranskat)abstract
- The relation between domain structures of recombinant monoclonal antibody fragments and their reaction kinetics was studied for the first time using a novel biosensor based on surface plasmon resonance technology. The association and dissociation rate constants of Fab, Fv and single domain (VH fragment) anti-lysozyme antibodies were determined and compared to the intact monoclonal antibody. Fab and Fv fragments showed similar reaction kinetics and had affinity constants of 6 X 109 M-1 and 25 X 109 M-1, respectively. The single domain antibody had significantly different reaction kinetics compared to the fragments consisting of paired heavy and light chain domains. The VH domain had both a higher dissociation and a lower association rate constant, which resulted in an affinity constant approximately 250 times lower than the Fab fragment. This rapid evaluation of antibody reaction kinetics should prove to be an important selection parameter when comparing antibody fragments for their utility in therapeutic or other applications.
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| 2. |
- 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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| 3. |
- Karlsson, Fredrik, et al.
(författare)
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Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function
- 2006
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 259:1, s. 81-88
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F+ and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F+. The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.
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| 4. |
- Karlsson, Fredrik, et al.
(författare)
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Genome-wide comparison of phage M13-infected vs. uninfected Escherichia coli
- 2005
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Ingår i: Canadian Journal of Microbiology. - 0008-4166. ; 51:1, s. 29-35
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Tidskriftsartikel (refereegranskat)abstract
- To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection.
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| 5. |
- Karlsson, Fredrik, et al.
(författare)
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The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains
- 2003
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Ingår i: Journal of Bacteriology. - 0021-9193. ; 185:8, s. 2628-2634
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Tidskriftsartikel (refereegranskat)abstract
- The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized
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| 6. |
- 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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| 7. |
- Malmborg Hager, Ann-Christin, et al.
(författare)
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Real time analysis of antibody-antigen reaction kinetics.
- 1992
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 35:6, s. 643-650
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Tidskriftsartikel (refereegranskat)abstract
- Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Tab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2–3×105 (m-1s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassays, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tnα antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.
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| 8. |
- Steinhauer, Cornelia, et al.
(författare)
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Single framework recombinant antibody fragments designed for protein chip applications
- 2002
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Ingår i: BioTechniques. - 0736-6205. ; :Suppl., s. 38-38
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Tidskriftsartikel (refereegranskat)abstract
- High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low, abundance protein. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused an recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragment, that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified Taken together designed scFv fragments based on a single molecular scaffold, readily accessible in Large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.
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