Margo Roberts - Academia.edu (original) (raw)
Papers by Margo Roberts
This article cites 28 articles, 18 of which can be accessed free
Biochemical and Biophysical Research Communications, Feb 1, 1988
Cette invention porte sur de nouvelles proteines chimeres recepteur co-stimulantes et sur des seq... more Cette invention porte sur de nouvelles proteines chimeres recepteur co-stimulantes et sur des sequences d'ADN les codant. Les recepteurs chimeres sont constitues d'au moins trois domaines dans une molecule a chaine unique: un domaine extracellulaire de liaison aux ligands, un domaine transmembranaire et un domaine cytoplasmique de signalisation de fonction effectrice co-stimulante, agissant en synergie avec un signal de fonction effectrice dans la cellule hote. Ces nouvelles proteines recepteur co-stimulantes comportent un second domaine cytoplasmique de signalisation de fonction effectrice. Cette invention traite, en outre, de cassettes d'expression contenant les acides nucleiques codant les nouveaux recepteurs chimeriques, de cellules hotes exprimant ces derniers ainsi que de techniques d'utilisation de ces recepteurs pour co-stimuler des fonctions effectrices dans les cellules ainsi que de techniques permettant d'utiliser des cellules exprimant les recepteurs ...
Cancer treatment and research, 1988
The role of estrogen in the growth of human breast cancers has been investigated at two levels. F... more The role of estrogen in the growth of human breast cancers has been investigated at two levels. First, we have studied the pS2 gene, whose transcription is stimulated by estrogen in the human breast cancer cell line, MCF-7. The pS2 gene product is a small, secreted polypeptide currently of unknown function, but with structural features similar to some growth factors. The expression of the pS2 gene has so far been detected only in MCF-7 cells and some breast cancer biopsies. Preliminary studies indicate that pS2 is a potential marker for hormone-dependent breast cancer. Ongoing studies will continue to focus on the implicated role of pS2 in the estrogen-mediated growth of breast cancers and its possible use as a marker for estrogen-dependent tumors. Second, we have analyzed the structure and function of the human ER. The receptor stimulates pS2 gene transcription by interacting with an ERE in the 5'-flanking region of that gene. A mutational analysis of the receptor protein has localized a DNA-binding domain, which determines target gene specificity, and a hormone-binding domain. These domains appear to be the only two regions of the receptor which are absolutely required for the transcription-activating function of the ER in transfection assays with reporter plasmids. The N-terminal region of the protein (regions A and B), which is necessary for increasing the efficiency of gene expression using the pS2 ERE, but not a vitellogenin ERE, may also play a role in transcription activation. Further progress in the characterization of the ER functional domains will require studies on target genes in a more physiological chromatin environment, as well as detailed physical analyses of receptor structure.
vivo survival of receptor-modified syngeneic T cells in patients with
L'invention concerne un nouveau systeme d'encapsidation retrovirale dans lequel des struc... more L'invention concerne un nouveau systeme d'encapsidation retrovirale dans lequel des structures d'encapsidation retrovirales illustrees par la figure 1 et des transcripts de vecteurs d'encapsidation sont produits a partir de plasmides a fort pouvoir d'expression par transfection dans des cellules humaines. Des titres eleves de retrovirus recombines sont produits dans les cellules infectees. Les procedes de l'invention consistent a utiliser les nouvelles structures retrovirales afin de transduire des cellules humaines primaires, y compris des lymphocytes B et des cellules souche hematopoietiques humaines a l'aide de genes etrangers mis en culture conjointement a rendements eleves. L'invention est utile dans la production rapide de surnageants viraux a titre eleve et pour transduire a l'aide de cellules a rendement eleve, lesquelles sont refractaires a la transduction par des moyens classiques.
Experimental Cell Research, 1990
To investigate the regulatory role of the conserved interferon consensus sequence (ICS) found in ... more To investigate the regulatory role of the conserved interferon consensus sequence (ICS) found in the 5' flanking region of HLA class I genes, we studied the binding of nuclear proteins to the ICS of HLA-A2 gene (ICS-A2) by the gel shift assay. Nuclear extracts from several human cell lines expressing different levels of surface class I molecules reveal an ICS-A2-protein complex of similar mobility, the amount of which varies in a cell type-dependent manner. In some cell lines, interferon-gamma treatment decreased the level of this complex. The overlapping enhancer A element also competes for the formation of this ICS-A2-protein complex. Footprinting and methylation interference analyses demonstrate that nuclear protein(s) protect specific sequences within the ICS-A2 element, suggesting that these protein(s) may represent interferon-sensitive transcription factors.
The Journal of Immunology
TCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities includ... more TCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities including cytolysis via the structurally related ζ- and γ-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR ζ-chain. Such ζ-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both ζ- and γ-CIRs to activate the cytolytic function of two distinct classes of FcγR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing ζ- and γ-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both ζ- and γ-based CIRs were capable of activating target-specific cytolysis by both NK cells ...
Blood, 2000
To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred sy... more To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in viv...
Journal of Virology, 1999
We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macr... more We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated ...
Blood, 2000
We have genetically engineered CD4+ and CD8+ T cells with human immunodeficiency virus (HIV) spec... more We have genetically engineered CD4+ and CD8+ T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4ζ, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (ζ) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 × 1010 autologous CD4ζ-modified CD4+and CD8+ T cells administered with (n = 11) or without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/μL and viral loads of at least 1000 copies/mL at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4ζ was detected in 1% to 3% of blood mononuclear cells at 8 weeks and 0.1% at 1 year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year....
Blood, 1994
We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was pr... more We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to...
This article cites 28 articles, 18 of which can be accessed free
Biochemical and Biophysical Research Communications, Feb 1, 1988
Cette invention porte sur de nouvelles proteines chimeres recepteur co-stimulantes et sur des seq... more Cette invention porte sur de nouvelles proteines chimeres recepteur co-stimulantes et sur des sequences d'ADN les codant. Les recepteurs chimeres sont constitues d'au moins trois domaines dans une molecule a chaine unique: un domaine extracellulaire de liaison aux ligands, un domaine transmembranaire et un domaine cytoplasmique de signalisation de fonction effectrice co-stimulante, agissant en synergie avec un signal de fonction effectrice dans la cellule hote. Ces nouvelles proteines recepteur co-stimulantes comportent un second domaine cytoplasmique de signalisation de fonction effectrice. Cette invention traite, en outre, de cassettes d'expression contenant les acides nucleiques codant les nouveaux recepteurs chimeriques, de cellules hotes exprimant ces derniers ainsi que de techniques d'utilisation de ces recepteurs pour co-stimuler des fonctions effectrices dans les cellules ainsi que de techniques permettant d'utiliser des cellules exprimant les recepteurs ...
Cancer treatment and research, 1988
The role of estrogen in the growth of human breast cancers has been investigated at two levels. F... more The role of estrogen in the growth of human breast cancers has been investigated at two levels. First, we have studied the pS2 gene, whose transcription is stimulated by estrogen in the human breast cancer cell line, MCF-7. The pS2 gene product is a small, secreted polypeptide currently of unknown function, but with structural features similar to some growth factors. The expression of the pS2 gene has so far been detected only in MCF-7 cells and some breast cancer biopsies. Preliminary studies indicate that pS2 is a potential marker for hormone-dependent breast cancer. Ongoing studies will continue to focus on the implicated role of pS2 in the estrogen-mediated growth of breast cancers and its possible use as a marker for estrogen-dependent tumors. Second, we have analyzed the structure and function of the human ER. The receptor stimulates pS2 gene transcription by interacting with an ERE in the 5'-flanking region of that gene. A mutational analysis of the receptor protein has localized a DNA-binding domain, which determines target gene specificity, and a hormone-binding domain. These domains appear to be the only two regions of the receptor which are absolutely required for the transcription-activating function of the ER in transfection assays with reporter plasmids. The N-terminal region of the protein (regions A and B), which is necessary for increasing the efficiency of gene expression using the pS2 ERE, but not a vitellogenin ERE, may also play a role in transcription activation. Further progress in the characterization of the ER functional domains will require studies on target genes in a more physiological chromatin environment, as well as detailed physical analyses of receptor structure.
vivo survival of receptor-modified syngeneic T cells in patients with
L'invention concerne un nouveau systeme d'encapsidation retrovirale dans lequel des struc... more L'invention concerne un nouveau systeme d'encapsidation retrovirale dans lequel des structures d'encapsidation retrovirales illustrees par la figure 1 et des transcripts de vecteurs d'encapsidation sont produits a partir de plasmides a fort pouvoir d'expression par transfection dans des cellules humaines. Des titres eleves de retrovirus recombines sont produits dans les cellules infectees. Les procedes de l'invention consistent a utiliser les nouvelles structures retrovirales afin de transduire des cellules humaines primaires, y compris des lymphocytes B et des cellules souche hematopoietiques humaines a l'aide de genes etrangers mis en culture conjointement a rendements eleves. L'invention est utile dans la production rapide de surnageants viraux a titre eleve et pour transduire a l'aide de cellules a rendement eleve, lesquelles sont refractaires a la transduction par des moyens classiques.
Experimental Cell Research, 1990
To investigate the regulatory role of the conserved interferon consensus sequence (ICS) found in ... more To investigate the regulatory role of the conserved interferon consensus sequence (ICS) found in the 5' flanking region of HLA class I genes, we studied the binding of nuclear proteins to the ICS of HLA-A2 gene (ICS-A2) by the gel shift assay. Nuclear extracts from several human cell lines expressing different levels of surface class I molecules reveal an ICS-A2-protein complex of similar mobility, the amount of which varies in a cell type-dependent manner. In some cell lines, interferon-gamma treatment decreased the level of this complex. The overlapping enhancer A element also competes for the formation of this ICS-A2-protein complex. Footprinting and methylation interference analyses demonstrate that nuclear protein(s) protect specific sequences within the ICS-A2 element, suggesting that these protein(s) may represent interferon-sensitive transcription factors.
The Journal of Immunology
TCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities includ... more TCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities including cytolysis via the structurally related ζ- and γ-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR ζ-chain. Such ζ-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both ζ- and γ-CIRs to activate the cytolytic function of two distinct classes of FcγR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing ζ- and γ-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both ζ- and γ-based CIRs were capable of activating target-specific cytolysis by both NK cells ...
Blood, 2000
To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred sy... more To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in viv...
Journal of Virology, 1999
We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macr... more We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated ...
Blood, 2000
We have genetically engineered CD4+ and CD8+ T cells with human immunodeficiency virus (HIV) spec... more We have genetically engineered CD4+ and CD8+ T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4ζ, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (ζ) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 × 1010 autologous CD4ζ-modified CD4+and CD8+ T cells administered with (n = 11) or without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/μL and viral loads of at least 1000 copies/mL at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4ζ was detected in 1% to 3% of blood mononuclear cells at 8 weeks and 0.1% at 1 year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year....
Blood, 1994
We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was pr... more We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to...