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Biomedical subjects

J A Titus

Publications and source records attributed to J A Titus.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1990

Targeting human T-lymphocytes with bispecific antibodies to react against human ovarian carcinoma cells growing in nu/nu mice.

In the present study we tested whether human T-cells from normal donors can be targeted against human ovarian carcinoma cells and block i.p. growth of an established tumor in immunodeficient mice. For targeting we used chemically cross-linked bispecific monoclonal antibodies (mAbs) reacting with CD3 on the T-cells and with cell-surface antigens selectively expressed by tumor cells. The tumor model consisted of mice given i.p. injections of a human ovarian carcinoma cell line, OVCAR-3, whose growth includes development of massive ascites. Peripheral blood lymphocytes from normal human donors were cultured overnight with 50-100 units/ml recombinant interleukin 2, coated with bispecific antibodies, and injected i.p. into mice 4-6 days after tumor inoculation, at which time tumor cells were established and growing in about 85% of the hosts. Tumor growth was assessed by the number of tumor cells, and in some tests by cell-free tumor antigen, recovered in peritoneal lavage fluid collected 15 days after tumor priming. Treatment with lymphocytes retargeted with bispecific mAbs, prepared with anti-CD3 and three different antitumor mAbs, 113F1, OVB-3, and MOv19, gave highly significant increases in percentages of mice without detectable tumor. Controls showed that the antitumor activity of retargeted lymphocytes did not result simply from antibody-dependent cellular cytotoxicity or from heteroconjugates reacting only with CD3 or with lymphocyte major histocompatibility complex determinants and tumor cells. These results show that targeted T-lymphocytes can significantly decrease the growth of an established tumor in a fashion specific for antigens expressed by the neoplastic cells.

Animals↗
PubMed1990

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.

Antigens, CD↗
PubMed1990

Improvement of glidobactin A production by Polyangium brachysporum.

Glidobactins A, B, and C are lipopeptide antitumor antibiotics produced by the gliding bacterium Polyangium brachysporum sp. nov. No. K481-B101. The production of glidobactin A was examined in shake flasks and laboratory fermentors. Medium screening and optimization led to approximately five fold increases in glidobactin A titers in shake flasks and a ten fold increase in titers in 40-l batch fermentations. Utilization of a stepped glucose feeding protocol resulted in glidobactin A titers of 1860 micrograms/ml after 144 h of fermentation.

Antibiotics, Antineoplastic↗
PubMed1990

Refocusing the immune system to react with human tumors by targeting human lymphocytes with bispecific antibodies.

Recent studies have established that crosslinking triggering sites on an immune cell, such as CD3 or FcR, to antigens on target cells with redirect the target-cell specificity of the immune cell. We are testing whether the human immune system can be refocused against human tumors with bifunctional antibodies produced by chemically crosslinking monoclonal antibodies against CD3 and tumor antigens with SPDP. The tumor model consists of immunodeficient (athymic) mice injected i.p. with a human ovarian cell line, OVCAR 3. Mice are treated 4-6 days later, after tumor growth has been established, with i.p. injections of human PBL from normal donors and bifunctional antibodies. Tumor growth is assessed by the amount of tumor cells and cell-free tumor antigen recovered in peritoneal lavage fluid 15 days after tumor injection. Relative to lymphocytes alone, bifunctional antibodies (including Fab preparations) increase the % of tumor-free mic from about 20 to 60%. A variety of controls show that both the bifunctional antibodies and the lymphocytes are required for the anti-tumor effect. Thus, heterocrosslinked antibodies can greatly enhance the anti-tumor activity in human PBL and may provide a new immunotherapeutic approach for treating human cancer.

Animals↗
PubMed1988

Expression of Fc gamma receptors on splenic T cells of mice infected with Plasmodium chabaudi adami.

In previous studies, it was shown that mice infected with Plasmodium chabaudi adami have a deficiency in their production of IgG1 immunoglobulin, suggesting isotype-specific immunoregulation. In order to examine this phenomenon in further detail the expression of Fc gamma receptors (Fc gamma R) on T cells obtained from mice infected with P. chabaudi was studied by flow cytometry. There was an increase in the number of splenic T cells which expressed Fc gamma R during infection. At the peak of the acute stage of infection (10-15 days) up to 40% of T cells were positive for Fc gamma R expression. These Fc gamma R were present on about 40% of both Lyt-2+ and L3T4+T cells. The isotype preference of these receptors on control Thy-1+ T cells is IgG1 greater than IgG2b greater than IgG2a as determined by an inhibition assay and fluorescence-activated cell sorter (FACS) analysis. However, 2 to 3 weeks after infection this pattern was altered such that IgG2b and IgG2a represented the major isotypes binding to the Fc gamma R of the L3T4+ T cell. At this stage of infection Fc gamma R on L3T4+ cells fail to bind IgG1. In the Lyt-2% T cells IgG1 and IgG2b remained the best inhibitors. These data suggest that there may be changes in Fc gamma R expression on T cells during infection reflected particularly in a decreased ability of IgG1 to bind to the Fc gamma R of L3T4+ cells.

Animals↗
PubMed1988

Targeting of cytotoxic cells against tumors with heterocrosslinked, bispecific antibodies.

Cytotoxic cells express specific receptors on their surfaces by which they distinguish altered or foreign cells from normal autologous cells. Recently, a method has been developed by which the natural recognition system of cytotoxic cells can be artificially manipulated, giving rise to cytotoxic cells of any desired specificity, including specificity against tumor and virally infected cells. The method for retargeting cytotoxic cells employs heterocrosslinked antibodies, in which one antibody is directed against the cytotoxic cell receptor (CCR) involved in lysis, while the second antibody is directed against a target cell structure, for example a tumor or viral antigen. By linking the CCR directly to the target cell, the heterocrosslinked antibodies promote the formation of effector: target conjugates and signal the cytotoxic cell to deliver a lethal hit. T cells can be targeted by heteroconjugates containing antibodies against components of the T cell receptor complex, e.g., Ti or CD3, while several types of antibody-dependent cellular cytotoxicity (ADCC) effector cells, including K/NK cells, macrophages, and neutrophils, are targeted using heteroconjugates containing antibodies against Fc gamma receptors. In peripheral blood from normal donors at least six types of targetable activities have been identified in vitro. In Winn type tumor neutralization assays in nude mice, targeted T and K cells can prevent the establishment of subcutaneous tumor at low effector: tumor ratios. Moreover, targeted human peripheral blood T cells cause the eradication of established intraperitoneal human ovarian carcinoma in nude mouse models. Targeted cytotoxic cells therefore hold great promise as a novel form of cancer immunotherapy in humans.

Animals↗
PubMed1987

Human K/natural killer cells targeted with hetero-cross-linked antibodies specifically lyse tumor cells in vitro and prevent tumor growth in vivo.

We have induced fresh peripheral blood K/natural killer cells to lyse a variety of target cells by coating them with anti-Fc gamma receptor (anti-Fc gamma R) (CD16) antibody hetero-cross-linked with anti-target cell antibody. The cytotoxic cell mediating this activity is different, as judged by depletion studies, from the CD3+, CD8+ T cell which is targeted by anti-CD3 cross-linked to anti-target cell antibody. Targeted K cell activity from some donors is enhanced by exposure to interleukin 2 but not interferon-gamma; other donors exhibit high amounts of this activity without stimulation. Specificity of lysis mediated by targeted K cells is dictated by the specificity of the anti-target cell antibody within the heteroconjugate, and bystander cells are not lysed by targeted K cells. Hetero-cross-linked antibodies containing anti-histocompatibility leukocyte antigen class I instead of anti-Fc gamma R (CD16) do not promote lysis, suggesting that the bridging of the target cell to Fc gamma R on the K cell is required to activate the lytic process. Lysis mediated by targeted K cells is much less inhibitable by polymerized IgG than is classical antibody-dependent cellular cytotoxicity. Fresh human melanoma cells are lysed specifically by K cells coated with anti-Fc gamma R (CD16) cross-linked to the 96.5 anti-melanoma antibody. In vivo, targeted K cells prevent tumor growth at low effector to target ratios in Winn-type tumor neutralization assays. Targeted K cells may therefore provide a new immunotherapeutic approach for the destruction of detrimental cells, such as tumor and virally infected cells.

Animals↗
PubMed1987

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice.

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.

Adenocarcinoma↗
PubMed1986

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody.

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.

Antibodies, Neoplasm↗
PubMed1986

Specific targeting of human peripheral blood T cells by heteroaggregates containing anti-T3 crosslinked to anti-target cell antibodies.

Antibody heteroaggregates have been used to render human peripheral blood T cells lytic for specified targets. The heteroaggregates contain anti-T3 covalently linked to antibodies against nominal target cell antigens. Such heteroaggregates bind target cells directly to T3 molecules on effector cells and trigger target cell lysis. Freshly prepared human PBL, when coated with anti-T3-containing heteroaggregates, are lytic without further stimulation, although brief exposure to crude lymphokine-containing supernatants or recombinant IL-2, but not recombinant IFN-gamma, enhances the activity. The effector cells are T8+, and when fully stimulated, their lytic activity approaches that of some cloned CTL. When T cells are treated with heteroaggregate, washed, and incubated at 37 degrees C in medium not containing heteroaggregate, they retain activity for at least 24 h. The results of this study suggest a strategy in which heteroaggregate-coated T cells could be used in vivo to mount a lytic response against pathogenic cells such as tumor cells or virus-infected cells.

Antibodies↗
PubMed1985

Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium.

A defined medium was developed for Streptomyces peucetius that optimally contained 0.5 mM magnesium, 1 mM phosphate, 75-125 mM glucose, 10 mM nitrate, and microelements. Poorer results were obtained with nitrite, aspartate, or ammonia as sole nitrogen sources. Other carbon sources which supported best growth and highest anthracycline titers were fructose, maltose, and soluble starch. In each case, substantial residual carbon remained at the end of 6 days, suggesting a lack of catabolite repression by the carbohydrate carbon sources on anthracycline biosynthesis. Studies involving limiting and nonlimiting concentrations of glucose supplemented with arabinose, a poorly utilizable carbon source, indicated that high carbon concentrations were not necessary for osmotic stabilization. Inorganic phosphate was found to have an inhibitory effect on anthracycline production. Furthermore, when cultures at early stages of anthracycline production were spiked with inorganic phosphate, a delay in further anthracycline production resulted until the added phosphate was depleted. A 10% inoculum of stationary phase cells yielded the best growth and most consistent anthracycline production. Spectrophotometric analyses at 495 nm of chloroform--methanol-extracted material were also found to be useful for the determination of total anthracyclines in culture extracts.

Antibiotics, Antineoplastic↗
PubMed1985

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens.

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.

Animals↗