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Biomedical subjects

M J Embleton

Publications and source records attributed to M J Embleton.

At least 19 recordsLinked to original sources
PubMed2005

Side population/ABCG2-positive cells represent a heterogeneous group of haemopoietic cells: implications for the use of adult stem cells in transplantation and plasticity protocols.

Murine side population (SP) cells may have an increased ability to engraft lethally irradiated mice and lack CD34 expression. Strategies using CD34 as a primary marker of haemopoietic stem cells may therefore result in the exclusion of a primitive stem cell population. The molecular basis for the murine SP phenotype has been attributed to the multidrug-resistance transporter ABCG2. This study aimed to investigate ABCG2 expression from a variety of human sources and investigate the relationship between ABCG2 expression, the SP phenotype, and expression of markers such as CD34 and CD133. SP cells were observed in different haemopoietic sources, but a significant increase in the number of SP cells was observed in PB following granulocyte colony-stimulating factor mobilisation. No direct correlation between the frequency of SP cells and the expression of ABCG2 was observed. SP cells were identified in both lineage-positive and lineage-negative population and ABCG2 expression was enriched in lineage-negative SP cells. Lineage-negative SP cells were devoid of CD34 expression but enriched for CD133. Subsequent analysis revealed that ABCG2 and CD133 are coexpressed. Together, these data suggest that the ABCG2 transporter is neither required nor responsible for the SP phenotpye in many human blood cells.

AC133 Antigen↗
PubMed2002

Targeted cytokine delivery to neuroblastoma.

The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.

Animals↗
PubMed2001

Anti-8-oxo-2'-deoxyguanosine phage antibodies: isolation, characterization, and relationship to disease states.

We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2'-deoxyguanosine (8-oxodG). One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light. In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg). The nucleotide sequence of the 175G V(H) gene segment was 98% homologous to the published V(H) sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE). Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G. The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity. 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1.

We have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment.

Amino Acid Sequence↗
PubMed2000

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Amino Acid Sequence↗
PubMed2000

Development of synthetic promoters for radiation-mediated gene therapy.

Exposure of cells to ionising radiation results in the activation of specific transcriptional control (CArG) elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. As part of a study investigating the potential use of these elements in radiation-controlled gene therapy vectors, we have incorporated their sequences into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. In vector-transfected MCF-7 breast adenocarcinoma cells, the synthetic promoter was more effective than the wild-type Egr1 counterpart in up-regulating expression of the reporter gene after exposure to a single 5 Gy dose, and equally effective as the wild-type in U87-MG glioma cells. The level of gene expression achieved using the synthetic promoter was dependent on the inducing radiation dose for both U87-MG and MCF-7 cells, being maximal at 3 Gy and decreasing at 5 and 10 Gy. Furthermore, induction could be repeated by additional radiation treatments. The latter indicates that up-regulation should be additive during fractionated radiotherapy schedules. To demonstrate the potential clinical benefit of such an approach, the synthetic promoters were also shown to drive expression of the herpes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclovir (GCV) when compared with cells treated with radiation alone. Our results demonstrate that the synthetic promoter is responsive to low doses of ionising radiation and therefore isolated CArG elements function as radiation-mediated transcriptional enhancers outside their normal sequence context. The continued development and optimisation of such radiation-responsive synthetic promoters is expected to make a valuable contribution to the development of future radiation-responsive vectors for cancer gene therapy.

Adenocarcinoma↗
PubMed2000

A radiation-controlled molecular switch for use in gene therapy of cancer.

Ionising radiation induces the expression of a number of radiation-responsive genes and there is current interest in exploiting this to regulate the expression of exogenous therapeutic genes in gene therapy strategies for cancer. However, the radiation-responsive promoters used in these approaches are often associated with low and transient levels of therapeutic gene expression. We describe here a novel radiation-triggered molecular switching device based on promoter elements from the radiation-responsive Egr-1 gene and the cre-LoxP site-specific recombination system of the P1 bacteriophage. Using this system, a single, minimally toxic dose of radiation induced cre-mediated excision of a lox-P flanked stop cassette in a silenced expression vector and this resulted in amplified levels of CMV-promoter-driven expression of the exogenous tumour-sensitising gene, HSV-tk. This strategy could be used in combination with targeted delivery and tumour-specific promoters to elicit the tumour-targeted and prolonged expression of a variety of tumour-sensitising genes and provide an unprecedented level of control and tumour selectivity.

Antiviral Agents↗
PubMed1997

In-cell assembly of scFv from human thyroid-infiltrating B cells.

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, to our knowledge, for the first time to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human thyroid tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, we obtained a unique 800-bp band corresponding in size to scFv fragments. We provide evidence that recombination between VH and VL genes occurred inside the same cell. This in-cell amplification and association procedure is a potentially useful tool for the study of autoantibody gene families and the VH/VL pairing that occurs during the autoimmune process.

B-Lymphocytes↗
PubMed1996

A CD66a-specific, activation-dependent epitope detected by recombinant human single chain fragments (scFvs) on CHO transfectants and activated granulocytes.

Antibodies to CD66 recognize at least five members (CD66a-e) of the carcinoembryonic antigen (CEA) family. Recombinant human single-chain Fv fragments (scFvs) that bind specifically to CD66a (biliary glycoprotein) were obtained from a naive human scFv library. The scFvs bound to the N-domain of CD66a on Chinese hamster ovary (CHO) transfectants but did not bind to freshly isolated peripheral granulocytes or to dimethylsulfoxide-treated HL-60 cells. In contrast, scFvs bound well to granulocytes that were short-term activated with N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate and to human HL-60 cells that were treated with all-trans-retinoic acid to induce granulocytic differentiation. Quantification of antigenic sites showed that the activation-dependent CD66a epitopes were expressed on nearly all of the CD66a molecules on CHO-biliary glycoprotein transfectants, but they were detected only on a portion of the molecules on activated polymorphonuclear neutrophils and differentiated HL-60 cells. Binding of CD66a scFvs to their neoepitopes on prestimulated PMNs induced respiratory burst, suggesting that CD66a is capable of delivering transmembrane signals in these cells.

Animals↗
PubMed1993

Human anti-self antibodies with high specificity from phage display libraries.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

Amino Acid Sequence↗
PubMed1992

In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.

Animals↗
PubMed1992

Monoclonal antibody targeting of methotrexate (MTX) against MTX-resistant tumour cell lines.

Several Methotrexate (MTX)-resistant sublines of the osteogenic sarcoma cell line 791T were derived by continuous selection in the presence of MTX and 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies including assays of the uptake and binding of [3H]MTX and fluoresceinated-MTX, determined that these sublines showed diminished MTX transport, and that none of them appeared to overproduce the MTX-target enzyme dihydrofolate reductase. Conjugates of the anti-791T monoclonal antibody 791T/36 linked to MTX via human serum albumin (HSA) were prepared by Dr M.C. Garnett. These were cytotoxic selectively for cells bearing the 791T/36-defined antigen (gp72), and were found to be as cytotoxic to most of the MTX-resistant 791T sublines as they were to parental 791T cells. Furthermore, an anti-MTX/anti-gp72 bispecific antibody 516 augmented the cytotoxicity of HSA-MTX conjugate to the MTX-resistant 791T variant R120 apparently as efficiently as for parental 791T cells. It is suggested that acquired drug resistance caused by deficient transport mechanisms may be partially or wholly overcome by targeting the drug to a readily-internalised cell surface antigen.

Antibodies, Monoclonal↗
PubMed1991

Efficacy and selectivity of monoclonal-antibody-targeted drugs and free methotrexate in fluorescence-labelled mixed tumour-cell monolayer cultures and multicellular spheroids.

Free methotrexate (MTX) and 2 monoclonal antibody (MAb)-MTX conjugates were tested against mixed human tumour-cell cultures, in which 2 cell lines of differing antigenicity or drug sensitivity, pre-labelled with fluorescent dyes, were added together in microtitre wells. Conjugates were selectively cytotoxic for cells bearing high concentrations of the relevant antigen, and MTX was preferentially cytotoxic for wild-type cells rather than MTX-resistant variants when tested on separate cultures. In mixed cultures these selectivities were substantially retained, although there was a varying tendency towards intermediate cytotoxicity for each cell line of the pair. MTX was cytotoxic for cell line 79 IT grown as multicellular spheroids, but a MAb-MTX conjugate and a MAb-RTA immunotoxin showed little cytotoxicity against spheroids at the highest concentrations tested, although they were highly effective against monolayer cells. Mixed spheroids could be formed efficiently from most cell lines, although in some cases cell distribution was non-random. In mixed spheroids prepared between wild-type and MTX-resistant 79 IT variants, relative MTX sensitivities conformed broadly to those seen in separate monolayer cultures. Fluorescence-labelling was a reliable method for determining cell behaviour under the above conditions. We conclude that (a) selectivity of therapeutic agents in mixed cultures was partially, but not completely, impaired compared to that observed in separate cultures, and (b) that low Mr drugs are effective against 3-dimensional tumour-cell structures but antibody-targeted conjugates are not.

Antibodies, Monoclonal↗
PubMed1991

Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action.

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

Ammonium Chloride↗
PubMed1991

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain.

A bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and ricin toxin A chain (RTA) was tested for its ability to target recombinant RTA (r-RTA) to CEA-expressing tumour cells, alone and in combination with ricin B chain (RTB). The antibody, 636 (Robins et al., 1990), induced significant RTA cytotoxicity against MKN45 gastric carcinoma cells which express high levels of CEA, using the r-RTA at a concentration below that known to be intrinsically cytotoxic. The addition of ricin toxin B chain (RTB) also potentiated cytotoxicity of r-RTA, and there was an additive increase in potentiation against CEA-positive cells when both RTB and 636 were included. The bispecific antibody restored potentiation by RTB after blocking of its binding site with excess galactose, and also the cytotoxic activity of whole ricin which had been blocked with galactose. It was concluded that the 636 bispecific antibody was highly effective in targeting the toxic moiety of the molecule to CEA-expressing cells, and allowed exploitation of the additional ability of the B chain to facilitate cellular incorporation. The facilitating function of the B chain was equally effective whether or not its lectin site was active.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of chemically induced neoplastic transformation in vitro by saturated fatty acids.

Syrian hamster embryo cells were treated for 2-4 days with 3-methylcholanthrene (MCA), and 28 days after application of the carcinogen they were exposed continuously to 12-O-tetradecanoylphorbol-13-acetate (TPA). Untreated cells, or cells treated with MCA or TPA only, usually became senescent around 6-8 weeks after plating and died, but those treated with both MCA and TPA became immortalised and underwent transformation to a phenotype capable of growth in soft agar. Transformation was inhibited by derivatives of stearic acid administered to the cells at various stages of the overall process. Some derivatives were active at lower concentrations than others, but none were selective for specific phases such as initiation or promotion, and it did not appear to matter whether the fatty acids were present throughout the assay or for only part of it. Their action seems to be associated with control of growth or differentiation in a relatively non-specific manner.

Animals↗