Biomedical subjects

H N Lode

Publications and source records attributed to H N Lode.

35 records · Page 2Linked to original sources

PubMed1998

Induction of persistent tumor-protective immunity in mice cured of established colon carcinoma metastases.

The induction of tumor-specific T-cell responses that are effective in eradicating disseminated tumors and in mounting a persistent tumor-protective immunity is one of the major goals of tumor immunotherapy. Here, we demonstrate that we achieved this goal by directing interleukin 2 (IL-2) to the tumor microenvironment of colon carcinoma metastases in syngeneic mice with a recombinant antibody-IL-2 fusion protein (huKS1/4-IL-2). Eradication of established pulmonary metastases is induced by a CD8+ T cell-mediated immune response, which can be transmitted to naive syngeneic severe combined immunodeficient mice by adoptive transfer of CD8+ T cells from immune animals. This immune response was followed by the induction of a long-lived immunity against challenge up to 5 months later with CT26-KSA or wild-type CT26 murine colon carcinoma cells in BALB/c mice. This memory immune response was confirmed by flow cytometric analyses of CD8+ T cells isolated from secondary lymphoid tissue that revealed a phenotypic profile typical of early memory T cells. This long-lived protective tumor immunity was successfully boosted to become optimally effective in all experimental animals by injections of noncurative doses of IL-2 fusion protein 4 days after challenge with tumor cells. Taken together, our results indicate that the huKS1/4-IL-2 fusion protein elicits a long-lived cellular memory immune response that can be amplified by additional applications of IL-2 fusion proteins. This approach could become useful for the treatment of colorectal carcinoma in an adjuvant setting, particularly in patients with minimal residual disease.

Adoptive Transfer↗

PubMed1998

Targeted therapy with a novel enediyene antibiotic calicheamicin theta(I)1 effectively suppresses growth and dissemination of liver metastases in a syngeneic model of murine neuroblastoma.

The suppression of metastases in malignant diseases is one of the major goals in targeted chemotherapy. This was achieved with an antibody drug conjugate between a novel, rationally designed enediyene antibiotic calicheamicin theta(I)1 of exceptionally high cytotoxic potency and an antiganglioside GD2 monoclonal antibody 14G2a. Effective suppression of hepatic metastases was demonstrated in a novel syngeneic model of murine neuroblastoma that simulates the situation in patients in terms of antigen heterogeneity and presence of the target antigen on normal tissues. Here, we describe the first successful use of calicheamicin theta(I)1 for targeted chemotherapy in a clinically relevant syngeneic metastasis model.

Aminoglycosides↗

PubMed1998

Ascorbic acid stimulates DOPA synthesis and tyrosine hydroxylase gene expression in the human neuroblastoma cell line SK-N-SH.

Ascorbic acid is well known to induce noradrenaline synthesis in sympathetic nervous cells. In a series of experiments we found that incubation of the neuroblastoma cell line SK-N-SH with ascorbic acid (100-500 microM) for 2 h results in a significantly enhanced synthesis of 3,4-dihydroxyphenylalanine (DOPA) and dopamine. Additionally, cDNA-polymerase chain reaction (cDNA-PCR) analysis of relative mRNA levels corresponding to the enzymes involved in catecholamine synthesis revealed a 3-fold increase of tyrosine hydroxylase gene expression after 5 days of incubation with ascorbic acid (200 microM), whereas expression of dopamine-beta-hydroxylase was found to be unaltered. In summary the data give evidence that ascorbic acid leads to enhanced DOPA production in SK-N-SH cells by two different mechanisms: at the metabolic level after short-term incubation and by increasing the tyrosine hydroxylase gene expression after long-term incubation. Based on these data we suppose that enhancement of DOPA synthesis by ascorbic acid may be useful in the treatment of early Parkinson's disease.

Ascorbic Acid↗

PubMed1998

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma.

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.

Amino Acid Sequence↗

PubMed1998

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy.

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

Animals↗

PubMed1998

Immunocytokines: a promising approach to cancer immunotherapy.

Recombinant antibody-cytokine fusion proteins are immunocytokines that achieve high cytokine concentrations in the tumor microenvironment and thereby effectively stimulate cellular immune responses against malignancies. The activation and expansion of immune effector cells, such as CD8+ T lymphocytes, by interleukin-2 immunocytokines resulted in the eradication of established pulmonary and hepatic metastases of murine melanoma and colorectal carcinoma in syngeneic mouse models. These immunocytokines were equally effective in eliminating established bone marrow and liver metastases of murine neuroblastoma by activating natural killer cells. The effective eradication of metastases by immunocytokines resulted in significant prolongation in life span of mice over that of controls receiving equivalent mixtures of antibody and interleukin-2, which failed to reduce the growth of disseminated metastases. Proof of concept was established, indicating that immunocytokine-induced activation and expansion of immune effector cells in the tumor microenvironment can effectively eradicate established tumor metastases. This promising new approach to cancer immunotherapy may lead to clinical applications that improve treatment of cancer patients with minimal residual disease in an adjuvant setting.

Animals↗

PubMed1998

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy.

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.

Animals↗

PubMed1997

Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow.

BACKGROUND: Advanced (stage 4) cases of neuroblastoma, a childhood cancer of the nervous system, are associated with high relapse rates, even after intensive chemotherapy, radiotherapy, and autologous bone marrow transplantation. Therefore, the use of monoclonal antibodies directed against the neuroblastoma tumor marker disialoganglioside GD2 (GD2), in combination with recombinant human interleukin 2 (rhIL-2), is under clinical investigation. We hypothesize that targeted cytokine immunotherapy with a recombinant anti-GD2 antibody-interleukin 2 fusion protein (ch14.18-IL-2) is superior to a combination of ch14.18 and rhIL-2. Our purpose was as follows: 1) to develop a syngeneic model for murine neuroblastoma that expresses GD2 and features both experimental and spontaneous metastases to bone marrow and liver, and 2) to assess anti-GD2-targeted IL-2 therapy in this mode. METHODS: A hybrid neuroblastoma cell line was used to generate the GD2-positive NXS2 cell line. Bone marrow and liver metastases were quantified by reverse transcription-polymerase chain reaction for tyrosine hydroxylase and by organ weight or counts of macroscopic tumor foci, respectively. All P values reported are two-sided. RESULTS: Injection of NXS2 cells resulted in disseminated bone marrow and liver metastases exhibiting stable, but heterogeneous expression of GD2. Treatment with fusion protein (10 microg/day for 6 days) effectively suppressed growth of both experimental and spontaneous metastases to bone marrow and liver (P<.001). In contrast, a mixture of rhIL-2 and ch14.18 at equivalent dose levels was inefficient. Only mice treated with ch14.18-IL-2 showed a twofold prolongation in life span (P<.001). CONCLUSION: Targeted IL-2 therapy with a ch14.18-IL-2 fusion protein elicits an effective antitumor response. Our data suggest that a study of ch14.18-IL-2 as an adjuvant treatment in patients with minimal residual disease may be of value.

Animals↗

PubMed1997

Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy.

A recombinant humanized antibody-interleukin 2 fusion protein (huKS1/4-IL-2) was used to direct IL-2 to the tumor microenvironment and elicit a T cell-mediated eradication of established pulmonary and hepatic CT26-KSA colon carcinoma metastases in syngeneic BALB/c mice. This antitumor effect was specific because a fusion protein, which was nonreactive with these tumor cells, failed to exert any such effect. The efficacy of the huKS1/4-IL-2 fusion protein in eliminating metastases was documented because mixtures of monoclonal antibody huKS1/4 with recombinant human IL-2 were ineffective and, at best, only partially reduced tumor load. Two lines of evidence indicated the eradication of metastases and the absence of minimal residual disease in animals treated with the fusion protein: first, the lack of detection of CT26-KSA cells by reverse transcription-PCR, which can detect one tumor cell in 10(6) liver cells; and second, the tripling of life span. The effector mechanism involved in this tumor eradication is dependent on T cells because the IL-2-directed therapy is ineffective in T cell-deficient SCID mice. The essential effector cells were further characterized as CD8+ T cells by in vivo depletion studies. Such T cells, isolated from tumor-bearing mice after fusion protein therapy, elicited MHC class I-restricted cytotoxicity in vitro against colon carcinoma target cells. Taken together, these data indicate that fusion protein-directed IL-2 therapy induces a T cell-dependent host immune response capable of eradicating established colon cancer metastases in an animal tumor model.

Animals↗

PubMed1997

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.

Antigens, CD34↗

PubMed1995

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of monoamine transporters in neuroblastoma cell lines: correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression.

Radiolabelled meta-iodobenzylguanidine (MIBG) has been widely used in scintigraphy and targeted radiotherapy in patients with neuroblastoma. Recently, it has been demonstrated that MIBG is incorporated into neuroblastoma cells by the noradrenaline transporter. In vitro experiments on SK-N-SH human neuroblastoma cells performed in the present study showed that uptake of MIBG is inhibited by noradrenaline, more so by dopamine and to a lesser extent, by serotonin, indicating that the respective transporters may also contribute to MIBG uptake. However, neither dopamine nor serotonin transporter gene expression was detected. Noradrenaline transporter gene expression was found in 4 of 6 investigated cell lines, which correlated with specific MIBG uptake. Furthermore, an inverse correlation of noradrenaline transporter and tyrosine hydroxylase gene expression, the key regulatory enzyme of catecholamine synthesis, was observed. These data show that MIBG is specifically incorporated only in neuroblastoma cells in which there is noradrenaline transporter gene expression. Furthermore, the catecholamine status in neuroblastoma cells is regulated by a coordinate expression of the key elements of catecholamine synthesis and reuptake systems.

3-Iodobenzylguanidine↗

PubMed1994

Ascorbic-acid-mediated iron release from cellular ferritin and its relation to the formation of DNA strand breaks in neuroblastoma cells.

Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

Ascorbic Acid↗

PubMed1993

Determination of ascorbic acid by isotachophoresis with regard to its potential in neuroblastoma therapy.

Analytical capillary isotachophoresis was used to determine ascorbic acid (AA) in different matrices (cell-free system, neuroblastoma cell extracts and urine). The system for purging bone marrow of neuroblastoma cells, including 6-hydroxydopamine (6-OHDA) and AA, was analysed with regard to the interaction of AA with 6-OHDA and its autoxidation product, hydrogen peroxide. Furthermore, analyses concerning the uptake of AA into neuroblastoma cells as well as its excretion in urine after uptake of large amounts were carried out.

Ascorbic Acid↗

PubMed1990

Release of iron from ferritin by 6-hydroxydopamine under aerobic and anaerobic conditions.

6-hydroxydopamine (6-OHDA) proved to be a very effective agent for iron release from ferritin. Iron release was enhanced in the presence of SOD, catalase and under anaerobic conditions. Ascorbic acid, a well known agent able to release iron from ferritin, increased the amount of released iron in more than an additive manner when used in combination with 6-OHDA. Similar to 6-OHDA, 6-hydroxydopa (Topa) and 1,2,4-benzenetriol were also able to release iron in large amounts; in contrast, catecholamines and other benzenediols were comparatively ineffective.

Aerobiosis↗

PubMedDate not supplied

Synthesis and preclinical characterization of a paclitaxel prodrug with improved antitumor activity and water solubility.

The development of novel chemotherapy strategies based on prodrugs remains a major challenge for effective treatment of malignancies. We tested the hypothesis that this can be achieved by a prodrug of paclitaxel where one biologically active center, represented by the C7 hydroxyl group, was blocked by a dihydroxypropyl side chain which can be hydrolytically cleaved by a pH-dependent, slow-release mechanism. The prodrug was synthesized by condensation of solketal chloroformate with the C7 hydroxyl group of paclitaxel followed by a ring-opening reaction to the dihydroxyl derivative. The cytotoxicity of the prodrug was similar to paclitaxel, when tested in vitro against a variety of human tumor cell lines. In vitro cell cycle analysis indicated that concentrations within the micromolar range of both drug and prodrug are required to induce sufficient G2M arrest. The hydrophilic paclitaxel prodrug proved to be more than 50-fold more water soluble than the parental drug and effectively converted to paclitaxel by pH dependent hydrolysis. Importantly, the prodrug could be used at a 3-fold higher maximum tolerated dose (MTD) and revealed a markedly improved antitumor activity in mice compared to paclitaxel. Taken together, our results demonstrate, that a hydrolytically activated paclitaxel prodrug exhibits greater water solubility and superior antitumor activity than the parental drug.

Animals↗

PubMedDate not supplied

Recombinant immunocytokines targeting the mouse transferrin receptor: construction and biological activities.

Localized cytokine therapies with recombinant monoclonal antibody-cytokine fusion proteins, designated immunocytokines, have become of increasing interest for tumor immunotherapy, since they direct immunomodulatory cytokines into the tumor microenvironment. To investigate their mechanisms of action in a variety of syngeneic tumor models, recombinant mouse cytokines IL2 and GM-CSF were engineered as fusion proteins to the carboxyl terminus of a chimeric rat/mouse antitransferrin receptor antibody, ch17217 and expressed in stable-transfected Chinese hamster ovary cells. The recombinant immunocytokines were readily purified by affinity chromatography and their binding characteristics were identical to those shown for the ch17217 antibody. The IL2 immunocytokine had an activity similar to recombinant mouse IL2, whereas the GM-CSF immunocytokine had enhanced cytokine activity relative to recombinant mouse GM-CSF. The clearance rates of ch17217 and the GM-CSF and IL2 immunocytokines were relatively similar with elimination phases (t1/2alpha) of 1.8 h and distribution phases (t1/2beta) of 83, 88, and 91 h, respectively. Both immunocytokines demonstrated effective antitumor activity by suppressing the growth of hepatic metastases of mouse neuroblastoma and pulmonary metastases of mouse colon carcinoma in syngeneic A/J and BALB/c mice, respectively. These results indicate that biologically effective IL2 and GM-CSF immunocytokines combine the targeting ability of an antitransferrin receptor monoclonal antibody with the immunomodulatory functions of each cytokine. Because of the universal expression of the transferrin receptor on mouse tumor cell lines, these constructs should prove useful to determine their efficacy in a wide variety of syngeneic mouse tumor models and to perform detailed studies of their modes of action.

Animals↗

PubMedDate not supplied

Ascorbic acid induces lipid peroxidation on neuroectodermal SK-N-LO cells with high endogenous ferritin content and loaded with MAb-ferritin immunoconjugates.

Neuroblasma-and other malignant cells often contain elevated amounts of iron-rich ferritin and H2O2 and may therefore be a potential target for pro-oxidative effects of ascorbic acid (AA), generating cytotoxic products e.g. by lipid peroxidation (LPO). The influence of H2O2 and iron, either in its free form or bound to ferritin, on AA induced LPO was first investigated using erythrocyte ghosts as a model system. Results of these experiments showed that AA induced LPO not only in the presence of free available iron but also in the presence of ferritin. Similarly, AA induced significant LPO in neuroectodermal SK-N-LO cells with elevated intracellular ferritin levels. These LPO promoting effects of ferritin in the presence of AA on SK-N-LO cells could also be observed using ferritin-immunoconjugates: for this purpose, ferritin was bound to human monoclonal antibodies (MAb-ferritin) recognizing ganglioside GD2 which is present in large quantities on cell surfaces of SK-N-LO and many neuroblastoma cells. We conclude that the pro-oxidative effects of AA could be exploited in the treatment of ferritin rich neuroblastoma in combination with chemotherapy or with MAb-ferritin immunoconjugates.

Antibodies, Monoclonal↗