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D M Segal

Publications and source records attributed to D M Segal.

At least 55 records · Page 3Linked to original sources
PubMed1994

CD44 is a cytotoxic triggering molecule in human peripheral blood NK cells.

Previous studies have shown that target cells that bind to CD44 adhesion molecules on cloned cytotoxic T cells are lysed by the CTL. To determine whether CD44 is also a cytotoxic trigger molecule in human PBL, we tested a bispecific Ab consisting of anti-CD44 Fab cross-linked to a Fab against a target cell Ag, in cytotoxicity assays using PBL as effectors. We found that PBL mediated lysis in the presence of the anti-CD44 bispecific Ab provided that the effector cells were stimulated with either IL-2 or IL-12. Cell fractionation experiments showed that CD44-directed cytolysis was mediated exclusively by CD56+ low buoyant density cells, mainly by NK (CD16+) cells, but also to a lesser extent by CD56+ T cells. CD44-directed cytolysis appeared in these subsets 24 to 48 h after addition of IL-2 and paralleled the acquisition of Ab-independent (LAK) activity; in contrast, these cells mediated Ab-dependent cellular cytotoxicity and CD3 redirected lysis before stimulation with IL-2. Unstimulated CD56+ cells uniformly expressed high levels of CD44 that increased modestly after incubation with IL-2. No changes in isoform expression in the extracellular domain of CD44 could be detected upon activation with the use of isoform-specific mAbs. Thus, lymphokine stimulation caused CD44 to become a cytotoxic trigger in subsets of PBL that mediated other forms of cytotoxicity and expressed CD44 before activation, suggesting that activation of these cells was accompanied by a coupling of CD44 to their lytic machinery.

Animals↗
PubMed1994

Mammalian expression and secretion of functional single-chain Fv molecules.

Single-chain Fv (sFv) proteins are genetically engineered molecules that consist of the two variable domains of an antibody connected by a polypeptide linker; they contain the antigen binding function of the parental protein in a single 30-kDa polypeptide chain. sFvs are usually produced in bacteria where they are insoluble and therefore require extensive refolding in vitro. In this report we followed the processing of three antibody sFvs (145-2C11 directed against murine CD3 epsilon chain, OKT9 against the human transferrin receptor, and U7.6 against dinitrophenyl groups) by transfected mammalian (COS-7) cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv with high efficiency. The sFvs contained an immunoglobulin light chain leader sequence, which directed them to the endoplasmic reticulum and allowed secretion into the medium. We found that the sFvs were secreted at different rates, with the rate-limiting step of secretion being their exit from the endoplasmic reticulum. We increased the secretion rate of one of the sFvs by introducing an asparagine-linked glycosylation site in FR1 of the heavy chain, and by using tunicamycin (an inhibitor of glycosylation) we found that glycosylated antibody sFvs were secreted faster than their nonglycosylated counterparts. All secreted sFvs specifically bound their antigens; where tested, at least 90% of the secreted sFv was functional. Therefore, mammalian cells can effectively fold and secrete sFv antibody and can provide a convenient system for testing and producing sFv proteins.

Animals↗
PubMed1994

Redirection of T cell-mediated cytotoxicity by a recombinant single-chain Fv molecule.

We have produced two single-chain Fv (sFv) proteins by bacterial periplasmic secretion, one sFv with specificity for the hapten DNP, and the other for the human transferrin receptor. After solubilization and refolding, we recovered several mg of active sFv per liter of bacterial culture. Each sFv bound to cells bearing the appropriate Ag and could be used to direct targeted cellular cytotoxicity. Targeting relied on a universal bispecific antibody designed to cross-link CD3 on the cytotoxic T cell with a peptide fused to the sFv carboxyl-terminus. The universal bispecific antibody was used in combination with the Ag-specific sFv to redirect human cytotoxic T cells to kill a variety of target cells. Such an approach has a number of advantages that may make it useful for the immunotherapy of cancer and other diseases.

Amino Acid Sequence↗
PubMed1994

Tyrosine-containing activation motif-dependent phagocytosis in mast cells.

FcR capable of triggering cell activation share with BCR and TCR a conserved intracytoplasmic tyrosine-containing activation motif (TAM). Besides cell activation, these receptors trigger other biologic responses, such as endocytosis of soluble ligands. Murine mast cells express two types of FcR that, when aggregated by antibodies and multivalent Ag, trigger the release of inflammatory mediators and cytokines. These are high affinity receptors for IgE (Fc epsilon RI) and low affinity receptors for IgG (Fc gamma RIII). They comprise each an IgE- or IgG-binding alpha-subunit and two TAM-containing subunits that associate with both receptors: a beta-subunit and a homodimeric gamma-subunit that can associate also with the other subunits of the TCR. Herein, we focused on biologic activities triggered in mast cells via the TAM of the gamma-subunits. Using rat basophilic leukemia (RBL) cells stably transfected with cDNA-encoding murine Fc gamma RIII alpha, we found that murine Fc gamma RIII trigger the phagocytosis of antibody-coated erythrocytes. Using RBL transfectants expressing Fc gamma RIII with a deletion of the intracytoplasmic domain of Fc gamma RIII alpha or chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the intracytoplasmic domain of Fc gamma RIII alpha, we showed that intracytoplasmic sequences of Fc gamma RIII alpha are neither necessary nor sufficient for Fc gamma RIII to trigger phagocytosis. Using RBL transfectants expressing chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the TAM-containing intracytoplasmic domain of murine Fc gamma RIII gamma, we demonstrated that intracytoplasmic sequences of Fc gamma RIII gamma are sufficient to trigger phagocytosis. Using RBL transfectants expressing the same Fc gamma RII-III gamma chimeras, in the TAM of which one, the other, or both tyrosine residues were mutated, we established that tyrosines in the TAM sequence are required for phagocytosis. Our results endow TAM gamma with previously unknown triggering capacities and Fc gamma RIII with new biologic properties.

Amino Acid Sequence↗
PubMed1994

Comparison of the ability of red cells sensitized with a bispecific anti-D x anti-Fc gamma RIII Fab fragment to activate human K cells and peritoneal macrophages through Fc gamma RIII.

The functional activity of Fc gamma RIII on human K cells from peripheral blood was compared with that of Fc gamma RIII on peritoneal macrophages (PM) separated from the waste material of patients undergoing peritoneal dialysis. Fc gamma R function was assessed in vitro using human monoclonal IgG1 anti-D (AB5) or a bispecific antibody comprising Fab fragments of AB5 chemically linked to Fab fragments of monoclonal anti-Fc gamma RIII, 3G8 (AB5 x 3G8). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, K cells mediated the lysis of papainized red cells sensitized with the AB5 x 3G8 bispecific antibody but not with AB5. In contrast, red cell lysis by PM was not promoted by AB5 x 3G8 although AB5 was active. However, this lysis, being inhibited by monomeric IgG, was presumably mediated via Fc gamma RI. AB5 x 3G8 also failed to promote the binding and phagocytosis of both papainized and native red cells by PM although 99% of red cells and over 90% of peritoneal cells bound the bispecific antibody. In marked contrast to K cells therefore, Fc gamma RIII on PM was unable to mediate functional interactions with red cells sensitized with anti-D x anti-Fc gamma RIII bispecific antibody.

Antibodies, Bispecific↗
PubMed1993

A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor.

A single-chain Fv construct of the 2B4 T-cell receptor has been made and expressed in Escherichia coli as bacterial inclusion bodies. After solubilization in 6 M guanidine hydrochloride and formation of mixed disulfides with glutathione, the protein was refolded by diluting out the denaturant and allowing intramolecular disulfide bridges to form by disulfide exchange. Approximately 65-100 mg of refolded protein was obtained from 1 liter of bacterial culture, an appreciable fraction of which was monomeric in nondenaturing solvents. This protein bound to three monoclonal antibodies specific for allotypic or idiotypic determinants on the native 2B4 variable region but did not bind several other anti-T-cell-receptor monoclonal antibodies that lacked such specificity. These experiments show that T-cell-receptor variable regions, like the V regions of antibodies, can form a well-behaved single-chain Fv molecule and provide large amounts of recombinant single-chain Fv T-cell receptor that can be used to study T-cell function.

Amino Acid Sequence↗
PubMed1993

Distinct intracytoplasmic sequences are required for endocytosis and phagocytosis via murine Fc gamma RII in mast cells.

In the present work, we studied the phagocytic and endocytic properties of murine Fc gamma RII in mast cells. Mouse mast cells express high-affinity receptors for monomeric IgE and three low-affinity receptors for complexed IgG: Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RIII. In previous studies we showed that, when aggregated by multivalent ligands, murine Fc gamma RIII, but not Fc gamma RII, triggers the release of inflammatory mediators and cytokines by mast cells. Upon Fc gamma R aggregation, mast cells not only release intracellular materials, they also internalize particulate and soluble immune complexes. We compared the ability of the two Fc gamma RII isoforms to trigger phagocytosis and endocytosis in RBL-2H3 cells stably transfected with cDNAs encoding wild-type, deleted, and tyrosine mutant Fc gamma RIIb1 or Fc gamma RIIb2. We found that Fc gamma RIIb2, but not Fc gamma RIIb1, triggered both phagocytosis and endocytosis. We identified distinct intracytoplasmic sequences necessary for Fc gamma RIIb2-mediated endocytosis and phagocytosis respectively, and we observed that two tyrosine residues, located in each of these sequences, are critical for endocytosis and/or phagocytosis. Our data indicate that the two internalization pathways diverge as early as signal transduction.

Amino Acid Sequence↗
PubMed1992

Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy.

We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

Animals↗
PubMed1992

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Animals↗
PubMed1992

Targeted cytokine production.

It has been well established that bispecific antibodies containing anti-T-cell receptor MAbs crosslinked to anti-tumor MAbs induce T cells to lyse tumor cells, as measured in a 51Cr-release assay. Such lysis requires direct attachment between target and cytotoxic cells and most probably involves the exocytosis of cytolytic substances into the cell:cell interface. In addition, targeted T cells mediate a second activity, the secretion into the medium of factors that can block the growth of bound tumor cells and unbound bystander cells. In order to test how targeted effector cells mediate anti-tumor effects in vivo, we are currently developing a totally syngeneic murine system in which murine T cells are targeted against mouse mammary tumors. The system allows us to treat both primary tumors and tumor transplants, using a mammary-tumor-virus antigen as the entity that is specifically recognized on the tumor cells.

Animals↗
PubMed1991

A nucleolar 2'-O-methyltransferase. Specificity and evidence for its role in the methylation of mouse 28 S precursor ribosomal RNA.

Methylation of ribose moieties appears to be an essential post-transcriptional event in ribosomal RNA maturation. Although the sites of ribose methylation have been identified, the components involved in the 2'-O-methylation of precursor ribosomal RNA in mammalian cells have not yet been elucidated. To investigate the involvement of a recently isolated nucleolar 2'-O-methyltransferase in this process, an in vitro synthesized 28 S rRNA transcript containing a unique tandem triple 2'-O-methylated ribose site was used as a substrate. Activity assays demonstrated that this transcript served as a substrate for the nucleolar 2'-O-methyltransferase. The distribution of incorporated methyl groups was determined by hydrolyzing the 2'-O-methylated transcript with RNase followed by chromatography of the digested products on an anion-exchange high performance liquid chromatography column. Results showed one unique RNase-resistant 2'-O-methylated product, a tetramer. The position of the tetrameric sequence in the 28 S rRNA transcript was determined using RNase protection analysis which mapped the methylations to a 20-nucleotide region spanning the unique tandem triple 2'-O-methylated ribose site in 28 S rRNA. To confirm the absolute specificity of methylation, direct sequence analysis was carried out on the tandem triple 2'-O-methylated tetramer. The sequence determined for the tetramer, AmGmCmA, corresponded exactly with that reported from in vivo studies. These findings demonstrate that the purified nucleolar 2'-O-methyltransferase can accurately methylate at a specific site of an in vitro derived preribosomal RNA transcript and support the proposed involvement of this nucleolar enzyme in ribosomal RNA maturation.

Animals↗
PubMed1991

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Amino Acid Sequence↗
PubMed1991

Human T-lymphocytes targeted against an established human ovarian carcinoma with a bispecific F(ab')2 antibody prolong host survival in a murine xenograft model.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.

Animals↗
PubMed1991

Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth.

We have compared the mechanisms by which human PBL targeted with bispecific antibodies either lyse tumor cells or block their growth in culture or in mice. We found that resting PBL were unable to mediate lysis, but were able to block tumor growth. Moreover, targeted PBL were unable to lyse bystander cells, whereas targeted PBL did block the growth of bystander tumor cells in culture and in nude mice. Supernatants from cultures of targeted PBL, or from PBL grown on anti-CD3-coated flasks, blocked the growth of tumor cells in the absence of added effector cells, and antibodies against TNF-alpha and IFN-gamma reversed the inhibition of tumor growth, but had no effect upon cytolysis mediated by targeted by PBL. Our results show that targeted human PBL mediate two different antitumor activities: lysis, which occurs rapidly and requires the direct attachment of the target cell to the cytotoxic cell, and tumor growth inhibition, which is mediated by cytokines released into the medium as a result of receptor cross-linking. The inhibition of bystander tumor growth in mice by targeted PBL suggests that factor release is sufficient to block tumor growth in vivo. Targeted factor release therefore provides a mechanism by which targeted PBL could block the growth of tumor cells in vivo that were not bound by the effector cells, but which were located in the vicinity of tumor cells that were bound.

Animals↗
PubMed1991

Processing fate of protein antigen attached to IgD or MHC molecules on normal B lymphocytes using heterocrosslinked bispecific antibodies.

We have studied the internalization, processing and presentation of hen egg lysozyme (HEL) attached to surface IgD (sIgD) or MHC molecules on normal murine B cells, using heterocrosslinked bispecific antibodies (HBA). Nearly all HEL attached to sIgD was internalized within one hour, with at least a portion rapidly entering a chloroquine-sensitive, acidic environment. Degradation and presentation of HEL to hybridoma T cells began several hours after internalization. Degraded HEL was found in the medium after about 6 hr incubation, but at no time were significant amounts of HEL peptides found within the cells. When HEL was attached to class I or class II MHC molecules, its rate of internalization was low. The fraction of antigen bound to MHC molecules that was inside the cell was always low, even at later stages of culture, but the internalized antigen was located in an acidic environment. Degradation and presentation of HEL internalized via MHC molecules followed internalization. No difference was observed in the processing fate of HEL attached to class I or class II MHC molecules. These results suggest that the rate limiting step in antigen processing and presentation is antigen degradation, when the antigen is bound to sIgD, and internalization when bound to MHC molecules. The slow and steady processing of bound or internalized antigen could provide a sustained presence of antigen on the B cell surface and enhance the potential for its presentation to T cells.

Animals↗
PubMed1991

Bispecific antibodies and targeted cellular cytotoxicity.

The Second International Conference on Bispecific Antibodies (BsAbs) and Targeted Cellular Cytotoxicity considered how targeted cytotoxicity can be used (1) to increase understanding of the general mechanisms of cellular cytotoxicity and (2) clinically in the treatment of cancer and infectious disease. BsAbs, the main mediators of targeted cellular cytotoxicity, can be made by chemical crosslinking, by fusing hybridoma cells and by molecular genetic approaches. BsAbs bind to target cells via one V region and trigger molecules such as T-cell receptors (TCRs) or FcR for IgG (Fc gamma R) on cytotoxic cells via their other V region. This linking of triggering structures to target cells induces target cell lysis and provides important clues to the signals used to elicit the lytic process.

Animals↗
PubMed1991

Activation of human T cells obtained pre- and post-interleukin-2 (IL-2) therapy by anti-CD3 monoclonal antibody plus IL-2: implications for combined in vivo treatment.

The role of activated T cells in the mediation of antitumor responses has been documented in several experimental models. In some of these, interleukin-2 (IL-2) has been used as a means to induce and expand the antitumor effects of the T cells. IL-2 has been tested in clinical trials for cancer treatment. Surprisingly, T cells appear to be inactivated by IL-2 in these clinical trials. T cells obtained from peripheral blood after IL-2 therapy showed decreased responses to mitogens and alloantigens, did not proliferate in vitro in response to IL-2, and did not mediate non-major histocompatibility complex-restricted cytotoxicity or targeted lysis in the presence of bispecific monoclonal antibodies. In this study, we present evidence that these post-IL-2 therapy T cells are not irreversibly inactivated; they can be activated in vitro by anti-CD3 monoclonal antibody together with IL-2 to upregulate the p55 component of the IL-2 receptor and proliferate. Nevertheless, following activation by anti-CD3 and IL-2, the level of targeted T-cell cytotoxicity mediated by the post-IL-2 therapy T cells was significantly lower than that by pre-IL-2 therapy T cells. Although in vivo treatment with IL-2 alone induces natural killer (NK) cells to mediate lymphokine-activated killer activity, these data suggest that the T-cell lytic function is inhibited by this treatment and only partially reversible by subsequent T-cell receptor activation using anti-CD3 mAb. Exposure of T cells to anti-CD3 mAb prior to in vivo IL-2 treatment generates T-cell lytic activity in vitro. These results, together with preclinical murine studies, suggest that a combined in vivo protocol of anti-CD3 mAb and IL-2, starting first with the anti-CD3 mAb, may cause activation of the T cells in addition to the activation of NK cells and thus warrant clinical testing.

Antibodies, Monoclonal↗