| 1. |
- Ohlin, M., et al.
(författare)
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Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope
- 1992
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 89:2, s. 290-295
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Tidskriftsartikel (refereegranskat)abstract
- Human MoAbs ofIgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.
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| 2. |
- Borrebaeck, Carl A K, et al.
(författare)
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Does endogenous glycosylation prevent the use of mouse monoclonal antibodies as cancer therapeutics?
- 1993
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Ingår i: Immunology Today. - 0167-5699. ; 14:10, s. 477-479
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies have many potential therapeutic benefits. However, when applied to humans, mouse monoclonal antibodies have several disadvantages. Here Carl Borrebaeck and colleagues describe a strategy to overcome the anti-Gal activity, thought to be one of the reasons why mouse mAbs have a limited half-life.
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| 3. |
- 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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| 4. |
- Chin, L. T., et al.
(författare)
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Site-directed primary in vitro immunization : Production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors
- 1994
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Ingår i: Immunology. - 0019-2805. ; 81:3, s. 428-434
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Tidskriftsartikel (refereegranskat)abstract
- The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T- cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287 467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significant frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.
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| 5. |
- De Cossío, Maria E Fernández, et al.
(författare)
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Human monoclonal antibodies against an epitope on the class 5c outer membrane protein common to many pathogenic strains of Neisseria meningitidis
- 1992
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 166:6, s. 1322-1328
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Tidskriftsartikel (refereegranskat)abstract
- Neisseria meningitidis is a causative agent of meningitis. Despite vaccination programs, it still causes a large number of deaths in young children. Early diagnosis followed by passive immunization with human monoclonal antibodies could be an approach to effective therapy. Peripheral blood lymphocytes from normal, healthy blood donors and from vaccinated individuals were immunized in vitro, using outer membrane proteins purified from A. meningitidis B:4:P1,15. The immunized human B cells were Epstein-Barr virus transformed and fused to a heteromyeloma. Several stable human hybridoma cell lines were established and two, secreting antibodies against the 31-kDa class 5c outer membrane protein, were characterized further. The human antibodies were of IgG1 and IgG3 isotypes, with κ light chains. The recognized epitope was commonly found among pathogenic strains of N. meningitidis; thus, these human monoclonal antibodies may be important in the evaluation of N. meningitidis infections.
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| 6. |
- Duenas, M., et al.
(författare)
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Intra- and extracellular expression of an scFv antibody fragment in E. coli : Effect of bacterial strains and pathway engineering using GroES/L chaperonins
- 1994
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Ingår i: BioTechniques. - 0736-6205. ; 16:3, s. 476-480
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter. For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector. The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding. Co-expression of chaperonin encoding plasmic pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein two-fold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.
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| 7. |
- Ifversen, P., et al.
(författare)
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Effect of cell-derived growth factors and cytokines on the clonal outgrowth of EBV-infected B cells and established lymphoblastoid cell lines
- 1993
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Ingår i: Human Antibodies and Hybridomas. - 0956-960X. ; 4:3, s. 115-123
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and is widely used as a tool for the establishment of B cell lines producing human monoclonal antibodies. However, because of low transformability, low clonability, and the inherent instability of EBV-infected B cells, valuable antibody-producing B cells are often lost during this procedure. We have here examined various cell-derived cytokines for their ability to enhance both the cellular outgrowth of newly infected B cells and the clonability of infected B cells and lymphoblastoid cell lines. Our results show that the murine thymoma cell line EL-4 is superior to peripheral blood mononuclear cells in both cellular outgrowth and cloning experiments, whereas monocyte-derived factors and monocyte cell lines were less capable than peripheral blood mononuclear cells in enhancing cellular outgrowth and cloning. Furthermore, the human T cell hybridoma cell line MP6 that secretes a B cell growth and differentiation factor, recently identified as an isoform of thioredoxin, is also capable of stimulating EBV-infected B cells and lymphoblastoid cell lines. Co-cultivation of EBV-infected B cells with MP6 cells significantly enhanced the cloning efficiency at the 1 cell/well level. The present results also suggest that one potential role of the MP6-derived thioredoxin could be the up regulation of IL-6 receptor expression in EBV-infected B cells.
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| 8. |
- 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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| 9. |
- Ohlin, M., et al.
(författare)
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Epstein-Barr virus-induced transformation of human B lymphocytes : the effect of l-leucyl-l-leucine methyl ester on inhibitory T cell populations
- 1992
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Ingår i: Immunology Letters. - 0165-2478. ; 34:3, s. 221-228
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus-mediated transformation of human B lymphocytes is inhibited by human T lymphocytes as well as by interferon-γ. Removal of the inhibitory cell populations is essential in order to achieve successful transformation in vitro. Cells with the capacity to inhibit outgrowth of lymphoblastoid cell lines can be removed by pretreatment of peripheral blood mononuclear cells with l-leucyl-l-leucine methyl ester. This treatment eliminates monocytes, NK-cells and a CD8+ T cell subpopulation. We now show that such treatment also has toxic effects on other human T cell populations. In addition, CD4+ and/or CD8+ lymphocytes are demonstrated to contain effector cell activities which inhibit outgrowth of EBV-transformed B cells. This inhibitory activity is abolished after treatment of peripheral blood mononuclear cells or purified CD4+ T cells with l-leucyl-l-leucine methyl ester. No evidence was found for a selective toxicity against any subset within the CD4+ or CD8+ T cell populations. However, the capacity of the treated cells, both peripheral blood mononuclear cells and purified CD4+ T lymphocytes, to produce mRNA encoding IFN-γ, a protein previously shown to downregulate outgrowth of EBV-transformed B cells, was selectively impaired. The results obtained suggest a role for CD4+ T cells to inhibit EBV-induced transformation of B cells.
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| 10. |
- Ohlin, Mats, et al.
(författare)
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Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies
- 1993
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Ingår i: Journal of Virology. - 0022-538X. ; 67:2, s. 703-710
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Tidskriftsartikel (refereegranskat)abstract
- The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-speciftc antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gpH6, potently neutralized the virus at 1 to 2 μg of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.
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