| 1. |
- Dueñas, Marta, et al.
(författare)
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Selection of phage displayed antibodies based on kinetic constants
- 1996
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 33:3, s. 279-285
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Tidskriftsartikel (refereegranskat)abstract
- The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3. In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their k(ass), k(diss) and K(a). All Feb fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1). We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies. Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure. Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions.
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| 2. |
- Ohlin, Mats, et al.
(författare)
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Characteristics of human antibody repertoires following active immune responses in vivo
- 1996
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 33:7-8, s. 583-592
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Tidskriftsartikel (refereegranskat)abstract
- Possibilities to develop human monoclonal antibody specificities have recently been much facilitated by improvements of human hybridoma technology but even more so by the emerging phage-display technique. However, until recently very little has been known about the characteristics at the molecular level of the induced, T cell-dependent human antibody response, frequently targeted by these techniques. Rather, the major part of available sequence information has been related to tumor-derived or autoreactive antibodies. We have now investigated high affinity, monospecific, human antibody repertoires as developed by hybridoma technology. The VH region gene usage among such in vivo-induced repertoires is in only some respects similar to that found in the total B cell population. A limited number of heavy-chain variable segment loci account for the majority of all induced antibodies. A particular VH gene locus (4-34) frequently employed by peripheral B cells and associated with autoreactive antibodies was rarely used by the induced repertoire. Furthermore, in particular antigen systems, V region usage differs from the total available repertoire, and heavy-chain CDR3 is generally longer among antibodies induced against foreign protein antigens than in the average B cell population. Light-chain gene usage is often restricted to just a few dominant genes frequently found among B cells in general. In comparison, variable regions derived by phage-display technology in some antigen systems display even longer heavy-chain CDR3 than hybridoma-derived antibodies. This technique also appears to select a different set of germline genes preferentially (both with respect to VH and JH) as compared to hybridoma technology. In summary, the T cell-dependent antibody response against foreign antigens appears to differ from the average circulating B cell in several ways, and thus does not seem to represent a random selection of the available repertoire.
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| 3. |
- Ohlin, Mats, et al.
(författare)
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Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity
- 1998
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Ingår i: Molecular Immunology. - 0161-5890. ; 35:4, s. 233-238
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Tidskriftsartikel (refereegranskat)abstract
- Antibody diversity, a molecular feature which allows these proteins to specifically interact with a diverse set of targets, is created at the genetic level by a variety of means. These include germline gene segment recombination, junctional diversity and single basepair (bp) substitution. We here demonstrate that a human high affinity antibody specific for an exogenous protein antigen carry three amino acid residues immediately adjacent to the first hypervariable loop of the heavy chain. These additional residues are shown not to be encoded by the germline repertoire. We also describe the characteristics of insertions and deletions, not found in any known germline sequence, within the first and second hypervariable loops of other previously described antibody-encoding genes. These findings demonstrate that insertions or deletions of entire codons provide yet another approach by which the human antibody repertoire is diversified in vivo. Since these major genetic modifications occur within or immediately adjacent to loops contributing to the antigen-binding site, they are likely to affect the binding properties of the mutated antibodies.
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| 4. |
- Ohlin, Mats, et al.
(författare)
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Light chain shuffling of a high affinity antibody results in a drift in epitope recognition
- 1996
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 33:1, s. 47-56
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Tidskriftsartikel (refereegranskat)abstract
- Human polyclonal and monoclonal antibodies against pathogens and toxins are potentially useful in the treatment of various diseases. A number of human monoclonal antibodies with protective capacity in vitro have been established by conventional hybridoma technology. However, with the development of phage-display technology, the possibility of specifically tailoring antigen-binding properties has improved substantially. We show here that the reactivity of a high affinity, virus-neutralizing human antibody against the AD-2 epitope of cytomegalovirus gB can be modified by introducing other Vkappa sequences together with the original VH sequence. The fine specificity, as determined by the requirement of particular amino acid residues in the epitope, is shifted in these new antibody fragments. It was also evident that the VH/Vkappa pairing was not promiscuous, since antibody fragments selected by phage display retained light chain sequences very similar to the original hybridoma-derived light chain, proving that a high affinity interaction was very dependent on a co-operativity between both variable domains. These findings show that phage display technology might modify the binding properties of pre-existing, high affinity antibodies.
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| 5. |
- Wingren, Christer, et al.
(författare)
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Antigen-binding sites dominate the surface properties of IgG antibodies
- 1995
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Ingår i: Molecular Immunology. - 1872-9142 .- 0161-5890. ; 32:11, s. 819-827
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Tidskriftsartikel (refereegranskat)abstract
- A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen-binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties.
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