Cytotoxic and antitumor activity of a recombinant tumor necrosis factor-B1(Fv) fusion protein on LeY antigen-expressing human cancer cells

We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the scFv region of monoclonal antibody (mAb) B1, an antibody that recognizes LeY antigen present on many human cancer cells. Our rationale for fusing the scFv to the COOH terminus of TNF was to diminish the binding of the fusion protein to TNF receptors because the COOH terminus of TNF is involved in binding, and thus to partially inactivate (detoxify) the molecule. The Fv region should then target and accumulate the fusion protein on cancer cells, which should compensate for the reduced binding affinity of the TNF moiety and lead to selective killing of TNF-sensitive antigen-expressing cancer cells. The fusion protein was expressed in Escherichia coli and found in insoluble inclusion bodies. After refolding and purification by anion exchange, Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to LeY antigen on cancer cells with the same affinity as B1(scFv) and B1(scFv) immunotoxins but with significantly lower affinity to the TNF receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to LeY antigen-expressing cancer cells that are sensitive to TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is antibody targeted and TNF mediated because it can be prevented (as shown on MCF-7 cells) by an antibody competing for LeY antigen binding and by an antibody that neutralizes TNF-alpha. TNF-B1(Fv) kills TNF-alpha-sensitive cells that do not express the target antigen only at much higher doses than TNF trimer, and it does not kill LeY-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regression of MCF-7 tumor xenografts in mice at doses that are not toxic to the mice. Thus, the reduced binding of the TNF moiety to TNF receptors, combined with binding of the B1(Fv) portion to LeY antigen, makes TNF-B1(Fv) an agent for selective killing of LeY-expressing TNF-sensitive cancer cells.     

Expression and activity of a recombinant chimeric protein composed of pokeweed antiviral protein and of human interleukin-2

Liv.100 is an improvised herbal formulation of Liv.52. Liv.52 is an important component of the ayurvedic system of medicine. This report highlights on the protective effect of Liv.52 and Liv.100 against in vitro peroxidation induced by hydrogen peroxide in rat liver homogenate. Addition of the two herbal formulations reduced the peroxidation effect of hydrogen peroxide in the dose- and time-dependent manner. The protective effect of the drugs is attributed to the enhanced supply of reduced glutathione that inhibit the deleterious process of lipid peroxidation. The results suggest on the antioxidant potential of Liv.52 and Liv.100.     

Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry

A sensitive assay was developed for the detection of neuroblastoma cell contamination in CD34+ selected and unseparated peripheral blood stem cells (PBSC) used for autologous transplantation in stage 4 neuroblastoma patients. Specifically, we established a non-radioactive nested cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expression combined with anti-disialoganglioside GD2 immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs correlated to cell line dependent basal TH gene expression levels and ranged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytochemistry was 1:10(5). We observed the highest PBSC contamination rate of 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and GD2 immunocytochemistry in combination. Furthermore, a clinically applied purging method, CD34+ selection by immunoabsorption (CD34+ purity 42.4%), was used on 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34+ selection and 56% (9/16) remained contaminated. A significant reduction of neuroblastoma cell contamination by CD34+ selection was not detectable, but the absolute amount of re-infused tumour cells was decreased due to 100-fold smaller cell counts of CD34+ selected grafts used for transplantation. 22 PBSC preparations were used for transplantation. A Kaplan-Meier analysis showed an event-free survival probability of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versus 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamination. Our data suggest that the combined use of TH nPCR and GD2 immunocytochemistry is optimal to detect contamination and monitor purging strategies.     

Characterization of the pulmonary lesions induced in rats by human recombinant interleukin-2

Histologic, electron microscopic, and immunohistochemical studies were made to analyze the structural features and the cellular composition of the pulmonary lesions produced in rats by the administration of interleukin-2 (IL-2). This agent induced pulmonary edema; thickening of alveolar septa; damage to endothelial cells in capillaries and venules, marked interstitial infiltration by cytotoxic T lymphocytes, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells (as demonstrated by cell counting in preparations stained immunohistochemically with peroxidase- and fluorochrome-labeled antibodies); and injury to bronchiolar and alveolar epithelial cells. Granular and agranular lymphocytes often were closely apposed to endothelial cells in capillaries and venules. Contacts between lymphocytes and type II alveolar epithelial cells also were observed. Damaged type II alveolar epithelial cells showed nuclear and cytoplasmic features that are considered indicative of apoptosis (confirmed by nick end labeling). Phagocytosis of apoptotic bodies by macrophages was occasionally found. These results support the concept that IL-2 induces cytotoxic vascular and parenchymal cell damage that is mediated by LAK cells and cytotoxic T lymphocytes, which make contacts with endothelial cells and type II alveolar epithelial cells. This damage appears to be exacerbated by the secondary release of a variety of vasoactive agents and inflammatory mediators.     

Neonatal lupus erythematosus: maternal IgG antibodies bind to a recombinant NH2-terminal fusion protein encoded by human alpha-fodrin cDNA

IgG antibodies to a cleavage product of alpha-fodrin (120 kDa alpha-fodrin) have recently been identified as organ-specific autoantibodies in primary Sj?gren's syndrome. In this study, we examined seroreactivity of mothers and infants with neonatal lupus erythematosus (NLE) to a recombinant NH2-terminal protein (120 kDa alpha-fodrin) of human alpha-fodrin. Serum samples were collected during the perinatal period in seven pregnancies of five mothers delivering offspring with NLE. Anti-120 kDa alpha-fodrin antibodies were identified by immunoblotting in six of seven perinatal maternal sera of offspring with NLE: one of two congenital heart block offspring and all five offspring with cutaneous NLE. These antibodies were placentally transmitted to infants. One of the five mothers had primary Sj?gren's syndrome, and four were asymptomatic. One asymptomatic mother did not demonstrate anti-120 kDa alpha-fodrin activity at the time of the first delivery of a congenital heart block infant, but was found to be positive at the time of subsequent delivery of a second child with cutaneous NLE. We propose that maternal antibodies to 120 kDa alpha-fodrin may be an additional serologic marker for the risk of NLE in anti-Ro/SS-A positive women.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Lipoprotein(a) inhibits lipopolysaccharide-induced tumor necrosis factor alpha production by human mononuclear cells

Lipoproteins can bind lipopolysaccharide (LPS) and decrease LPS-stimulated cytokine production. Lipoprotein(a) [Lp(a)] was as potent as low-density lipoproteins (LDL) in inhibiting LPS-stimulated tumor necrosis factor synthesis by human mononuclear cells. The kinetics of LPS inhibition by Lp(a) was similar to that of LDL. This suggests that circulating Lp(a) may be an important factor determining the amplitude of the response to LPS in humans.     

Soluble tumor necrosis factor receptor (p75) fusion protein (ENBREL) as a therapy for rheumatoid arthritis

Soluble tumor necrosis factor (TNF) receptor fusion protein (p75) (Enbrel) is a reversible inhibitor of the biologic effects of TNF. Enbrel has been shown in placebo-controlled trials to significantly improve the signs and symptoms of rheumatoid arthritis. Clinical trials are now in progress to assess the safety and efficacy of Enbrel in combination with methotrexate in refractory rheumatoid arthritis along with trials to compare Enbrel to methotrexate in patients with early rheumatoid arthritis.     

Differential sensitivity of human myeloma cell lines and normal bone marrow colony forming cells to a recombinant diphtheria toxin-interleukin 6 fusion protein

The cytotoxicity of a recombinant interleukin 6 (IL-6)-diphtheria toxin (DT) fusion protein towards human myeloma cell lines was investigated. DAB389-IL-6 inhibited protein synthesis and methylcellulose colony formation by U266 myeloma cells. In the clonogenic assay, the fusion protein approached the level of cytotoxicity achieved by native DT. The specificity of killing by DAB389-IL-6 was demonstrated by inhibition of cytotoxicity by a molar excess of free rhIL-6. The effect of DAB389-IL-6 on colony formation by six OCI-My cell lines was assessed. Similar to U266 cells, colony growth by the OCI-My 5 and -My 2 cell lines was inhibited in a simple dose dependent manner. However, a biphasic effect was observed for the IL-6 dependent OCI-My 4 cells; DAB389-IL-6 stimulated colony formation at low (< or = 10(-11) M) concentrations, yet was inhibitory at higher doses. Three other cell lines whose growth was not altered by IL-6 were relatively unaffected by DAB389-IL-6, despite their sensitivity to native DT. Flow cytometric analysis for IL-6 receptor expression using phycoerythrin-conjugated IL-6 demonstrated specific binding sites on both DAB389-IL-6 sensitive and certain insensitive cell lines, suggesting that other factors in addition to the expression of IL-6 receptors are involved in killing by the fusion toxin. Despite evidence for a role of IL-6 in myeloid cell development, normal bone marrow was insensitive to the IL-6 fusion toxin. In cultures containing both normal bone marrow and U266 cells DAB389-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth. From these experiments we conclude that DAB389-IL-6 is specifically cytotoxic towards a subset of IL-6-responsive human myeloma cell lines and may be useful, in some cases, in the selective elimination of tumour cells from mixed populations of normal and malignant cells.     

In vivo delivery of interleukin-4 by a recombinant vaccinia virus prevents tumor development in mice

To study the immunotherapeutic potential of interleukin-4 (IL-4) delivered in vivo via a recombinant vaccinia virus, a thymidine kinase-negative (TK-) vaccinia virus that expressed the murine IL-4 gene (VV1/IL-4) was constructed. When mice were inoculated with 10(7) plaque-forming units (pfu) of VV1/IL-4 subcutaneously (s.c.), 10(5) pfu/cm2 were found in skin, and smaller numbers in liver and kidney between 1 and 7 days after infection; few viral pfu were found in spleen and lung, or in any organ after intravenous infection. This suggested that recombinant vaccinia viruses might be most efficient at delivery of cytokine genes to the skin. Because IL-4 has recently been found to have potent anti-tumor activity, the effect of recombinant virus infection on the development of s.c. tumors was studied. A single s.c. inoculation with VV1/IL-4 delayed the development of NCTC 2472 tumors, but when VV1/IL-4 was inoculated s.c. weekly for 8 weeks, tumor development was completely prevented in 93% of mice. Similarly, the development of M-3 melanoma tumors was also prevented by weekly s.c. inoculations of VV1/IL-4. About 40% of mice treated with control VV2/beta gal by the same regimen also failed to develop tumors. Weekly virus treatment did not prevent NCTC 2472 tumor development in athymic nu/nu mice, suggesting that mature T cells are required for expression of VV1/IL-4 induced antitumor activity. Thus, recombinant vaccinia viruses may be especially well suited for convenient therapeutic delivery of immunomodulator genes to skin-related sites.     

Activation of LA-N-2 cell phospholipase D by amyloid beta protein (25-35)

Amyloid beta protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25-35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and APP (25-35) was calcium-independent. The AbetaP (25-35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AbetaP (25-35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AbetaP (25-35). Only NBQX was effective with an IC50 of 75 microM for AbetaP (25-35) and quisqualate. Activation of phospholipase D by AbetaP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AbetaP (25-35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AbetaP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AbetaP (25-35).     

Activation of human T and B cells by rabbit anti-human beta 2-microglobulin

Rabbit anti-human beta 2-microglobulin (anti-beta 2m) increased the highest DNA synthesis in unseparated lymphocytes or artificially composed mixtures enriched in T and B cells. In enriched T and B cells no or low stimulation was seen. The maximal response by IgG anti-beta 2m was seen in proportions of enriched T/B cells, being 3:1 for blood lymphocytes and 1:1 for spleen cells, which are the same as the physiological proportions of T and B cells in these lymphoid organs. Whereas unseparated lymphocytes gave a peak response on day 3, enriched B cells had a peak response on day 6. Fab anti-beta 2m did not activate enriched T cells but increased DNA synthesis in enriched B cells to about the same extent as unseparated lymphocytes and mixtures of enriched T and B cells. The proportion of sheep erythrocyte rosette-forming cells (E-RFC) decreased after stimulation by anti-beta 2m and increased after stimulation by phytohaemagglutinin. However, as revealed by autoradiography, a proportion of lymphocytes activated by anti-beta 2m were E-RFC and this proportion increased with increasing stimulation by anti-beta 2m. DNA synthesis induced by anti-beta 2m was unchanged for spleen cells and slightly decreased for blood lymphocytes when phagocytic cells were removed by iron treatment. Supernatants from lymphocytes activated by anti-beta 2m only induced low DNA synthesis in enriched T or B cells. Experiments with mitomycin treated cells indicate that cooperation and close contact between T and B cells are needed for activation by IgG anti-beta 2m. T cells are needed for B cell activation by anti-beta 2m and B cells are required for T cell activation to occur.     

Expression of a voltage-dependent potassium current precedes fusion of human muscle satellite cells (myoblasts)

Using the whole-cell recording patch clamp technique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (IKdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed IKdr. In contrast, IKdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differentiating medium. We believe that IKdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following reasons: 1) after the transfer in differentiating medium, cultures of SC in which the expression of IKdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; 2) in the latter "naive" SC, IKdr was expressed during the first day in differentiating medium, before SC began to fuse; 3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express IKdr; 4) TEA, at a concentration of 3 mM, reduces the amplitude of IKdr by 55% and the fusion index by 55-67%.     

Suppression of in vitro IgM rheumatoid factor production by diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) in patients with refractory rheumatoid arthritis

OBJECTIVE: To investigate the effect of diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) on in vitro synthesis of immunoglobulin and rheumatoid factor (RF) in patients with severe refractory rheumatoid arthritis (RA) enrolled in a phase II, double blind, placebo controlled study. METHODS: Anticoagulated venous blood samples were obtained before (Day 1) and after (Day 28) intravenous infusion of either DAB 486IL-2 at 0.075 mg/kg/day (12 patients) or saline placebo (10 patients) on Days 1-5. Peripheral blood leukocytes (PBL) were prepared by density gradient centrifugation, cultured in the presence and absence of pokeweed mitogen (PWM) for one week, and culture supernatants assayed for immunoglobulins and IgM RF by ELISA. RESULTS: Compared to placebo treated patients, PWM induced IgM RF synthesis by PBL decreased after treatment with DAB 486IL-2 (p = 0.043). However, there was no apparent correlation with clinical improvement. PWM induced IgM, IgA, and IgG synthesis also tended to decrease, although the changes did not attain statistical significance. In contrast, PWM induced IgM RF, IgM, IgA, and IgG synthesis by PBL from patients treated with placebo tended to increase during the observation period. Spontaneous immunoglobulin and IgM RF production by PBL from either the DAB 486IL-2 or placebo patients remained stable. CONCLUSION: These observations raise the possibility that DAB 486IL-2 may diminish B cell function either directly or indirectly through effects on T cell function, but the change may not correspond to clinical response.     

Hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) in the perfused rat liver

The hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) were investigated in perfused rat liver to clarify the mechanism of hepatic clearance which is a major contributor to the rapid clearance of rhIL-11 in vivo. We analyzed the disposition characteristics of [(111)In]-labeled rhIL-11 using a single-pass constant infusion mode at different concentrations of rhIL-11. The venous outflow rapidly reached a steady-state condition at every concentration. Liver extraction ratio at steady-state (Ess) was decreased with increase in the concentration, suggesting that there is a saturable interaction between the liver cells and rhIL-11 molecule. Cellular localization experiments demonstrated that rhIL-11 was taken up by both liver parenchymal and nonparenchymal cells depending on their surface area, suggesting that this uptake was mediated by electrostatic interaction due to cationic charges in the cytokine.     

Activation of antigen-induced lymphocyte proliferation by interleukin-15 without the mitogenic effect of interleukin-2 that may induce human immunodeficiency virus-1 expression

The newly identified cytokine, IL-15 enhanced antigen-induced proliferation of PBMC obtained from HIV-1-seropositive subjects. When compared to IL-2 which enhanced both spontaneous and antigen-induced lymphocyte proliferative responses, IL-15 rarely increased spontaneous lymphocyte proliferation. Additionally, in cultures of lymphocytes obtained from 15 HIV-1-infected patients with < 300 circulating CD4- lymphocytes/microliter IL-15 induced significant HIV-1 expression (46, 21, and 71 pg/ml) in only 3 of 15 experiments and IL-2 induced significant HIV-1 expression (range 16- > 5000 pg/ml) in 11 of 15 experiments (P < 0.01, Fischer's exact test). Simultaneous assays of cytokine-induced spontaneous lymphocyte proliferation and HIV-1 expression revealed similar dose-response relationships for induction of HIV-1 and lymphocyte proliferation by IL-2. Thus, IL-15 helps to correct the impaired proliferative response of CD4+ lymphocytes from HIV-1-infected persons without the mitogenic effect of IL-2 that also may induce HIV-1 expression.     

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.