Intrathymic function of the human cortical epithelial cell surface antigen gp200-MR6: single-chain antibodies to evolutionarily conserved determinants disrupt mouse thymus development.
(9/1180)
The mouse monoclonal antibody MR6 recognizes a 200 000 MW protein (gp200-MR6), which is expressed highly on human thymic cortical epithelial cells. The antigen is also expressed on some epithelial tumours and we have previously shown that MR6 inhibits the proliferation of the colon carcinoma cell lines HT29. However, the role of this molecule in the thymus is not known. In order to generate reagents that could be used in murine thymic functional studies we isolated antibodies specific to human gp200-MR6, using a phage display library expressing single-chain (sFv) antibodies. Three independent clones were isolated by panning with purified protein and their specificity was confirmed by immunohistochemistry, Western blotting and flow cytometry. In addition to human thymus, these phage antibodies also recognized the homologous antigen in mouse, pig and other species. Expressed as soluble sFv one of these clones inhibited the proliferation of HT29 cells and a mouse thymic epithelial cell line, suggesting that this antibody exhibits similar functional activity to MR6. In fetal thymic organ culture, thymocytes recovered from thymic lobes cultured in the presence of this sFv, were reduced in number fivefold compared with the control and the majority remained at the double-negative stage of development. These data indicate that gp200-MR6 plays an important role in thymocyte development. In addition, this is the first report to demonstrate that specific sFv can be used to study, and alter, thymic development. This work also highlights the advantage of phage antibody technology in selecting such reagents for functional assays. (+info)
Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor.
(10/1180)
Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. (+info)
Further characterization of IgA in chicken serum and secretions with evidence of a possible analogue of mammalian secretory component.
(11/1180)
Immunochemical studies of the intestinal secretory immune system of the chicken have led to further characterization of IgA in bile, intestinal contents and serum. A component was detected in late Sephadex G-200 fractions of caecal and intestinal contents which showed partial identity with bile, intestinal and a high molecular weight fraction of serum IgA. This component showed similar sedimentation characteristics to bovine serum albumin in sucrose density gradients, a fast electrophoretic mobility on polyacrylamide gel and is a possible analogue of mammalian secretory component (SC). Fractionation of serum from birds affected with infectious synovitis revealed two moleculare classes of IgA. Comparative double diffusion studies produced a reaction of complete identity between bile IgA and high molecular weight serum IgA (15S) and partial identity with low molecular weight serum IgA (7S), suggesting a lack of an SC determinant on the latter. A spur of partial identity between 15S and 7S serum IgA was also observed. Although no direct structural homology with mammalian or human IgA could be demonstrated by immunological cross-reactivity, the similarities of molecular characteristics, particularly emphasized by the presence of a secretory component, favour a functional analogy between the secretory immune system of the fowl and mammalian species. (+info)
In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.
(12/1180)
For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis. (+info)
Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.
(13/1180)
HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection. (+info)
Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody.
(14/1180)
Tenascin-C exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. Here, we report a human antibody fragment, TN11, derived from a phage library with high affinity for the spliced repeat C and demonstrate that this repeat is undetectable in normal adult tissues, barely detectable or undetectable in breast, lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade astrocytoma (grade III and glioblastoma), especially around vascular structures and proliferating cells. The antibody appears to have potential for development of a therapeutic agent for patients with high grade astrocytoma. (+info)
Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.
(15/1180)
The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection. (+info)
Antigen recognition by conformational selection.
(16/1180)
Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an 'induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an 'induced fit' requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer. (+info)