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T Logtenberg

Publications and source records attributed to T Logtenberg.

At least 37 records · Page 2Linked to original sources
PubMed1999

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments.

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

Animals↗
PubMed1999

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.

Animals↗
PubMed1999

New strategies in anti-vascular cancer therapy.

Angiogenesis is a critical step in the progression of tumours from dormancy to a clinical relevant cancer. Inhibition of this process is one of the most promising new anti-cancer strategies. To develop new drugs that interfere with the cascade of events required for the formation of new blood vessels, insight into this process is essential. Here, we discuss the molecular basis of angiogenesis and the concepts of vascular targeting. Furthermore new strategies will be discussed to discover surface markers on endothelial cells that confer sufficient specificity for targeted intervention in the tumour vasculature.

Antigens, Neoplasm↗
PubMed1999

Dissecting the human peripheral B-cell compartment with phage display-derived antibodies.

Previously we have employed a large semisynthetic phage antibody display library, in combination with subtractive selection by flow cytometry to isolate phage antibodies specific for subpopulations of leucocytes. In this study, human tonsillar B cells were incubated with the phage library and IgD- CD38- memory B lymphocytes and attached phage antibodies were selected by cell sorting. In a panel of 17 monoclonal phage antibodies obtained, five displayed binding to cells of multiple haematopoietic lineages or broadly reacted with B-lineage cells. Immunofluorescent, immunohistochemical and biochemical studies permitted the characterization of the target molecules recognized by these phage antibodies. The remaining 12 antibodies displayed restricted binding to small subpopulations of peripheral human B cells. These results show that subtractive selections with phage antibody display libraries in combination with flow cytometry yield antibodies that bind to differentially expressed molecules on closely related cell populations, and can be used as a tool in a variety of assays.

Antibodies, Monoclonal↗
PubMed1998

Cloning, chromosal mapping, and tissue expression of the gene encoding the human Eph-family kinase ligand ephrin-A2.

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.

Adult↗
PubMed1998

Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein.

We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

Antibodies, Viral↗
PubMed1997

Regulated expression of the Eph-related receptor tyrosine kinase Hek11 in early human B lymphopoiesis.

Members of the large Eph family of receptor tyrosine kinases (RTKs) display temporally and spatially restricted expression patterns during embryogenesis, suggesting a role in various developmental processes. We have begun to investigate the expression of members of this receptor family during human hematopoiesis, in particular B lymphopoiesis. Expression of Eph RTKs in cells of the B-lymphoid lineage was assessed by using degenerate oligonucleotide primers based on stretches of conserved nucleic acid sequences in members of the Eph family. First, the content of Eph-family RTKs was assessed in freshly sorted fetal bone marrow pro-B cells. This population was found to harbor transcripts of the Hek8 and Hek11 members of this gene family. Subsequent analysis of expression of these genes in B cells representing various differentiation and ontogenic stages showed that the Hek8 transcript is constitutively present in all fetal and adult B-lineage cells, with high levels of expression in peripheral blood B cells. In contrast, the Hek11 transcript was exclusively found in fetal bone marrow pro-B cells and pre-B cells, but not in more mature fetal B-lineage cells. All adult B-lineage cells, from early pro-B cells to end-stage plasma cells, lacked Hek11 transcripts. The developmentally regulated expression of Hek11 during fetal B lymphopoiesis suggests a role for this gene in pre/pro-B cell expansion and/or differentiation and defines a difference in progenitor B cell populations isolated from fetal versus adult human bone marrow.

Adult↗
PubMed1997

TCR expression in human fetal intestine and identification of an early T cell receptor beta-chain transcript.

TCR expression by human fetal intestinal intraepithelial lymphocytes (ilELs) and intestinal lamina propria lymphocytes was analyzed to address whether T cell development occurs in human fetal intestine, the diversity of human fetal iIELs, and whether human fetal iIELs may contribute to the adult iIEL repertoire. ilELs and intestinal lamina propria lymphocytes from second trimester human fetal intestine were analyzed for TCR-alphabeta transcripts. Rearranged TCR-alpha transcripts were undetectable at 14 wk in the intraepithelial lymphocytes (IELs), whereas multiple TCR-beta transcripts were found at this stage. The TCR-alpha repertoire remained restricted relative to TCR-beta at later stages, and the IEL repertoire was restricted relative to the lamina propria lymphocytes at all stages. A previously reported T early alpha message was the major transcript from the TCR-alpha locus early in gestation. A previously undescribed TCR-beta transcript initiating upstream of the Dbeta1 locus and spliced to Cbeta1 or Cbeta2 was also identified and may represent a T early beta message. These results provide evidence for ongoing TCR gene rearrangement in human fetal intestine and suggest that transcription from the TCR-beta locus initiates with a T early beta transcript. The TCR-alpha repertoire (and hence the repertoire of potentially functional IELs) was limited through the second trimester.

Base Sequence↗
PubMed1997

Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.

Animals↗
PubMed1997

Bactericidal antibody recognition of a PorA epitope of Neisseria meningitidis: crystal structure of a Fab fragment in complex with a fluorescein-conjugated peptide.

Class 1 outer membrane protein PorA of Neisseria meningitidis is a vaccine candidate against bacterial meningitis. Antibodies against PorA are able to induce complement-mediated bacterial killing and thereby play an important role in protection against meningococcal disease. Bactericidal antibodies are all directed against variable regions VR1 and VR2 of the PorA sequence, corresponding to loops 1 and 4 of a two-dimensional topology model of the porin with eight extracellular loops. We have determined the crystal structure to 2.6 A resolution of the Fab fragment of bactericidal antibody MN12H2 against meningococcal PorA in complex with a linear fluorescein-conjugated peptide TKDTNNNL derived from the VR2 sequence of sero-subtype P1.7,16 (residues 180-187) from meningococcal strain H44/76. The peptide folds deeply into the binding cavity of the Fab molecule in a type I beta-turn, with the minimal P1.16 epitope DTNNN virtually completely buried. The structure reveals H-bonds and van der Waals interactions with all minimal epitope residues and one essential salt bridge between Asp-182 of the peptide and His-31 of the MN12H2 light chain. The key components of the recognition of PorA epitope P1.16 by bactericidal antibody MN12H2 correspond well with available thermodynamic data from binding studies. Furthermore, they indicate the structural basis of an increased endemic incidence of infection by group B meningococci in England and Wales since 1981 associated with the occurrence of an Neisseria meningitidis escape mutant (strain-MC58). The observed three-dimensional conformation of the peptide provides a rationale for the development of a synthetic peptide vaccine against meningococcal disease.

Amino Acid Sequence↗
PubMed1996

Biosynthetically lipid-modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes.

A human anti-CD22 single chain (sc) Fv antibody fragment from a synthetic phage antibody display library was biosynthetically lipid-tagged by using Escherichia coli lipoprotein sequences. The purified anti-CD22 scFv lipoprotein was incorporated into liposomes by detergent dilution. Anti-CD22 immunoliposomes were shown to bind specifically in a dose- and time-dependent manner to CD22+ cell lines and CD22+ B-lymphocytes present in freshly isolated samples of blood mononuclear cells. The immunoliposomes were demonstrated to accumulate in intracellular compartments. Biosynthetically lipid-tagged human scFv antibody fragments isolated from phage display libraries may facilitate the construction of immunoliposomes with improved properties.

Antigens, CD↗
PubMed1996

Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library.

This report describes the construction of leucine zipper-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers. A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library. Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments. The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts. The bivalent (scFv)2 fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis. Fos and Jun homodimer (scFv)2 antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv)2 Fos-Jun heterodimers. These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity.

Amino Acid Sequence↗
PubMed1995

Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

Abortion, Spontaneous↗
PubMed1995

Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions.

We have constructed a large (3.6 x 10(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline VH genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens. Western blotting assays and immunofluorescent analysis of cells by flow cytometry. The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.

Amino Acid Sequence↗
PubMed1995

Selective expansion and long-term culture of human Schwann cells from sural nerve biopsies.

Fragments of sural nerve biopsy specimens were cultured in the presence of the supernatant of lymphokine-activated killer cells, resulting in the selective outgrowth of cells with bipolar or tripolar morphology, reminiscent of that of Schwann cells. Immunofluorescent staining with antibodies to the S-100 protein, the low-affinity nerve growth factor receptor, and the surface Thy-1 antigen confirmed that these cultures contained more than 99% Schwann cells and no detectable fibroblasts. The mitotic activity of Schwann cells was measured by bromodeoxyuridine labeling, and was increased when the cells were grown in medium with lymphokine-activated killer cell supernatant compared with medium without this supernatant. In the presence of lymphokine-activated killer cell supernatant, Schwann cells could be maintained in continuous culture for a minimum of 8 months.

Biopsy↗