Antibody-dependent Complement-mediated Cytotoxicity Research Articles

Augmentation of humoral and cell mediated immune responses by Thujone

Thujone, a naturally occurring monoterpene, was found to enhance the total WBC count, bone marrow cellularity, number of α-esterase positive cells, number of plaque forming cells in spleen and circulating antibody titer in Balb/c mice (1mg/kg body weight, intraperitoneally for 5days). Thujone treatment enhanced proliferation of splenocytes and thymocytes, both in the presence and absence of specific mitogens. Administration of Thujone was found to stimulate the cell-mediated immunological response in normal and tumor bearing Balb/c mice. A significant enhancement in natural killer (NK) cell mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) in both normal as well as tumor-bearing animals was observed after the administration of Thujone. Production of cytokines such as IL-2 and IFN-γ was significantly enhanced by the administration of Thujone. The stimulatory effect of Thujone on cytotoxic T lymphocyte (CTL) generation was determined by Winn's neutralization assay using CTL sensitive EL4 thymoma cells. Thujone treatment showed a significant increase in CTL production in both the in vivo and in vitro models, as indicated by a significant increase in the life span of tumor bearing animals. All these results indicate that administration of Thujone could enhance the immune response of mice. There was a significant reduction in solid tumor development, mediated by the presence of alert immune responses during Thujone administration.

An Antibody-based Multifaceted Approach Targeting the Human Transferrin Receptor for the Treatment of B-cell Malignancies

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

Effect of Thuja occidentalis and its polysaccharide on cell-mediated immune responses and cytokine levels of metastatic tumor-bearing animals

Context: Tumor microenvironment induces an active immune tolerance and escapes immune surveillance. In order to achieve an effective antitumor immune response, appropriately activated immune cells should maintain their antitumor activity to overcome the immune suppressive tumor microenvironment.Objectives: This study focuses on the effect of Thuja occidentalis L. (Cupressaceae) extract and its polysaccharide (TPS) on cell-mediated immune response (CMI) in metastasis bearing mice.Materials and methods: Metastasis was induced by injecting B16F-10 melanoma cells in mice through the tail vein and effector mechanisms of CMI was studied by analyzing cytotoxic T-lymphocyte (CTL) activity, natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC). The effect of T. occidentalis and TPS on pro-inflammatory cytokines and tissue inhibitor matrix metalloproteinases (TIMP) levels were also analyzed.Results and discussion: Administration of T. occidentalis and TPS enhanced the NK cell activity, ADCC and ACC much earlier than the control tumor-bearing animals. T. occidentalis and TPS were also found to decrease the elevated level of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, GM-CSF and tumor necrosis factor (TNF)-α in the serum of metastatic tumor-bearing animals. The level of antitumor factors such as IL-2 and TIMP was elevated by the treatment with T. occidentalis and TPS in the serum, which was lowered in the untreated tumor-bearing animals.Conclusion: This study clearly suggests that T. occidentalis and TPS effectively stimulate cell-mediated immune system and decrease pro-inflammatory cytokines, thereby inhibiting metastasis of tumor cells.

Abstract 3622: An antibody-based multifaceted approach targeting the human transferrin receptor for the treatment of multiple myeloma

Abstract We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, CD71) that demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is due to its ability to decrease TfR1 levels leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc gamma receptors and the complement component C1q, suggesting that is capable of eliciting Fc-mediated effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Additionally, in two disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant anti-tumor activity including long-term survival. Interestingly, the parental antibody without avidin (ch128.1) also shows remarkable in vivo anti-cancer activity despite its lack of in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the Long-Term Cell-Initiating Culture (LTC-IC) assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2011-3622

Augmentation of cellular immune response byIpomoea obscuraand Ipobscurine alkaloid attenuates tumor growth in mice

The immune status of the host plays a crucial role in controling the process of carcinogenesis. General or selective activation of various immunocompetent cells and their secretory function to maintain a healthy immune status may help in cancer prophylaxis, as well as therapy. The present study focused on the effect of Ipomoea obscura and Ipobscurine on cell-mediated immune response. In this study we evaluated the effect of I.obscura and an indole alkaloid fraction from I. obscura on effector mechanisms of cell-mediated immune response by analyzing cytotoxic T lymphocyte (CTL) activity, natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC). The effect of I. obscura and Ipobscurine on interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels was also analyzed. In the in vitro and in vivo systems, I. obscura and Ipobscurine treatment augmented cell-mediated immune response by enhancing the killing activity of CTL and NK cells from splenocytes in normal as well as tumor-bearing mice. ADCC and ACC were also enhanced significantly in both normal and tumor-bearing animals after drug administration, compared with untreated control. Administration of I. obscura and Ipobscurine significantly enhanced the production of IL-2 and IFN-γ in normal as well as tumor-bearing animals. This study reveals that both I. obscura and Ipobscurine have the potential to augment immune response through the enhanced secretion of IL-2 and IFN-γ by T cells and thereby inhibit tumor growth and as an alternative medicine for cancer treatment.

Ofatumumab for newly diagnosed and relapsed/refractory chronic lymphocytic leukemia

Monoclonal antibodies have become an increasingly utilized treatment option for many hematological malignancies, including chronic lymphocytic leukemia (CLL). Ofatumumab is a second-generation fully human anti-CD20 monoclonal antibody that binds to the small extracellular loop of CD20, thereby producing complement-dependent cell lysis and antibody-mediated cell cytotoxicity in cells expressing CD20. Ofatumumab has shown efficacy in the treatment for relapsed or refractory CLL. This success has resulted in the recent US FDA approval of ofatumumab for the treatment of CLL in patients who are refractory to fludarabine and alemtuzumab-based regimens. Major side effects of ofatumumab include infusion reactions, neutropenia and increased risk for infection. This article provides an overview of the current data supporting the use of ofatumumab for CLL and projects the future role of ofatumumab as monotherapy and combination therapy.

Effect of vernolide-A, a sesquiterpene lactone from Vernonia cinerea L., on cell-mediated immune response in B16F-10 metastatic melanoma-bearing mice

One of the major reasons for the rapid progression of cancers is the ability of tumor cells to escape from the immune surveillance mechanism of the body. Modulation of immune responses is highly relevant in tumor cell destruction. Effect of vernolide-A on the cell-mediated immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of vernolide-A enhanced natural killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared with the metastatic tumor-bearing control. Administration of vernolide-A significantly enhanced the production of interleukin (IL)-2 and interferon-gamma (IFN-γ) in metastatic tumor-bearing animals. In addition, vernolide-A significantly down-regulated the serum levels of proinflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and granulocyte–macrophage colony-stimulating factor (GM-CSF) during metastasis. All these results demonstrate that vernolide-A could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.

Abstract B38: Silencing of mortalin, the mitochondrial hsp70, as a novel anticancer treatment strategy

Abstract A handful of monoclonal antibodies have recently been introduced in treatment of hematopoietic malignant neoplasms and solid tumors. The complement system, an important arm of the immune system, is activated following anti-tumor antibody treatment. Complement proteins assemble to form channel-like pores called MAC (Membrane Attack Complexes) that inflict damage to target cells. Unfortunately, cancer cells have many defensive strategies against antibody-dependent complement-mediated cytotoxicity, including MAC shedding by exocytosis. Our recent results have implicated mortalin, the mitochondrial heat shock protein 70, in the removal of the MAC from the cell surface. Mortalin is known to have tumor promoting and anti-apoptotic effects. Our research hypothesis states that mortalin is a vital protector of tumor cells from immune damage. In order to examine this hypothesis, we set up two mouse models of cancer, of K562 erythroleukemia cells in nude mice and EL4 T-lymphoma cells in C57BL/6 mice. Mortalin siRNA was administered to tumor bearing mice with or without concomitant tumor directed antibodies. First, in vitro experiments showed that mortalin siRNA acts synergistically with anti-cancer antibodies in lysis of tumor cells. In vitro mortalin silencing in tumor cells by siRNA and then injection of the cells into mice demonstrated reduced tumor growth, relative to cells pretreated with scrambled siRNA. Mortalin silencing in vivo by intratumoral or intravenous injection of mortalin siRNA led to a significant decrease in tumor growth rate. Furthermore, mortalin siRNA also reduced tumor blood supply and caused tumor necrosis. Growth rate of EL4 and K562 tumors was also significantly inhibited by injection of the mortalin inhibitor MKT-077. Mortalin inhibition in tumors may thus be regarded as a novel adjuvant treatment to immunotherapy. Citation Information: Cancer Res 2009;69(23 Suppl):B38.

Complement-dependent antibody mediated cytotoxicity (C'AMC) in patients with newly diagnosed insulin-dependent diabetes mellitus.

Serum activities of complement-dependent antibody mediated cytotoxicity (C'AMC) were determined in 36 consecutive patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). The sequential exposure of 51Cr labeled neonatal rat islet cells to patient serum and rabbit complement revealed the presence of C'AMC in 28 IDDM subjects. The C'AMC titres ranged between 1:4 and 1:512 and were not related to the C'AMC activity of a given sample as measured at a standard dilution (1:4). In comparison to the clinical characteristics of 21 IDDM patients with negative C'AMC, higher C'AMC titres (greater than or equal to 1:32) were associated with a lower mean age at diagnosis of IDDM (12.2 +/- 2.1 vs. 19.0 +/- 2.3 years; p less than 0.05), with a higher frequency of infections up to 6 months prior to diagnosis (6 out of 11 vs. 3 out of 21 patients; p less than 0.05) and, although statistically not significant, a preponderance of female sex together with a decreased frequency of HLA-DR4. In contrast, fasting C-peptides levels, HLA-DR3 antigen frequency and Coxsackie B1-6 virus antibody titres were not related to the C'AMC titres. It is concluded that (1) C'AMC titration is superior to the detection of initial C'AMC levels for evaluating the strength of the complement-dependent humoral immune response towards islet cell surface (auto)antigen(s), and (2) infectious agents may be involved in eliciting a C'AMC response.

The effects of sera obtained from children with insulin-dependent diabetes mellitus on cultivated islet cells: cytotoxicity and insulin release.

Sera were obtained from 24 patients with newly-diagnosed insulin-dependent diabetes mellitus (IDDM) and 14 children with a high risk of diabetes. The influence of the decomplementated sera on basal and stimulated insulin secretion was studied in a mixed culture of newborn rat islet cells. In addition, complement-dependent antibody-mediated cytotoxicity (C'AMC) was measured by 51Cr-release from pre-labelled islet cells. Incubation of the islet cells with sera from ten IDDM patients did not affect the basal insulin release. Sera from other children with IDDM (n = 14) either significantly increased (n = 7) or inhibited (n = 7) basal IRI secretion was compared with the sera of control donors. Nearly half of the sera from the high-risk children was found to be insulin-stimulating. Preincubation of islet cells with sera from IDDM children caused a significant decrease of insulin response to 16.5 mM glucose plus 5 mM theophylline (P less than 0.001). Sera from the high-risk children did influence the response of pancreatic cells to secretagogues. C'AMC was found in 45% of the patients with IDDM and in 33% of the high-risk children, however, there was no correlation between C'AMC and serum effect upon basal insulin secretion. These results suggest the presence of B-cytotropic factors in serum from children with IDDM or with a risk of diabetes. Opposite effects of different sera on insulin secretion may reflect the variety of pathogenetic mechanisms involved in islet cell destruction.

Antibody-Based Therapy for Solid Tumors

Monoclonal antibodies have emerged as a class of novel oncology therapeutics. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the immune system differentiate these biologics from traditional small molecule anticancer drugs. In this review, we focus on 4 antibodies approved for clinical use in treating solid tumors, trastuzumab (Herceptin) for breast cancer, bevacizumab (Avastin) for colorectal cancer and non-small cell lung cancer, cetuximab (Erbitux) for colorectal cancer and head and neck cancer, and panitumumab (Vectibix) for colorectal cancer. The anticancer effects of these antibodies derive from blockade of growth factor/receptor interaction and/or down-regulation of oncogenic proteins (eg, growth factor receptors) on the tumor cell surface, and for some of these antibodies from the ability to elicit effector mechanisms of the immune system, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. The mechanism behind each antibody, the registration trials for their approved indications, and emerging indications are the focus of this article. We also review clinical considerations including commonly observed and antibody-related side effects, and dosing schedules. In addition, perspectives on challenges and opportunities of oncology antibody clinical development, antibody engineering, and the use of pharmacogenomics are presented.

Hepatitis C Virus (HCV)-Induced Immunoglobulin Hypermutation Reduces the Affinity and Neutralizing Activities of Antibodies against HCV Envelope Protein

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.

Effect of Naturally Occurring Triterpenoids Ursolic Acid and Glycyrrhizic Acid on the Cell-Mediated Immune Responses of Metastatic Tumor-Bearing Animals

Effect of terpenoids, ursolic acid and glycyrrhizic acid on the cell-mediated immune response was studied in metastatic tumor-bearing C57BL/6 mice. Intraperitoneal administration of ursolic acid and glycyrrhizic acid (50 μmoles/Kg body wt for 5 consecutive days) was found to produce increased natural killer cell activity (NK-activity) in metastatic tumor-bearing animals. In the glycyrrhizic acid- and ursolic acid-treated groups the peak activity was observed on the 4th day (60.2% and 43% cell lysis respectively). Whereas in control animals the maximum cell lysis was obtained only on 16th day (25% cell lysis). Administration of terpenoids clearly enhanced the antibody dependent cell mediated cytotoxicity (ADCC). In tumor-bearing control animals maximum cell lysis was obtained only on the 16th day (20% cell lysis). But in the case of glycyrrhizic acid- and ursolic acid-treated groups, the maximum lysis was obtained on the 12th day. and it was 47% and 32.5% cell lysis, respectively. Intraperitoneal administration of these 2 terpenoids was also found to enhance antibody-dependent complement-mediated cytotoxicity (ACC) in metastatic tumor-bearing animals. The elevated level of GM-CSF in tumor-alone treated control animals (37.9 ± 1.1 pg/ml) was reduced by the treatment with glycyrrhizic acid (20.3 pg/ml) and ursolic acid (22.5 pg/ml). The highly elevated level of IL-6 (370.1 pg/ml) in control animals was also reduced by the treatment of glycyrrhizic acid (313 pg/ml) and ursolic acid (299 pg/ml). The level of IL-2 was enhanced by the treatment with glycyrrhizic acid (37.9 pg/ml) and ursolic acid (35.9) compared to untreated tumor-bearing control animals (24.9 pg/ml).

Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

A Novel Cytotoxic Mechanism by Leukemia-Specific Antibody in Immunotherapy with Leukemia Cell-Derived Heat Shock Protein 70.

Introduction: Tumor-derived heat shock proteins (HSPs), which bind the tumor-specific antigenic peptides, are good application for cancer vaccine. We previously reported that immunotherapy using leukemia cell-derived HSPs against leukemia cell in mice prolonged survival by leukemia-specific cellular immune responses through CD8 + cytotoxic T-cell (Sato et al. Blood, 2001; Iuchi et al. Int J Hematol, 2006). We also indicated that CD4+ as well as CD8+ T-cell is indispensable for the survival prolongation (Sato et al. Blood, 2001), suggesting that humoral immune response by CD4+ T-cell also contributes to eradicate leukemia cells. Contributions of humoral immune responses, including tumor-specific antibodies or cytotoxic activities, in anti-tumor immunity induced by tumor-derived HSP-based immunotherapy remain unclear. We therefore investigated humoral immune responses against leukemia cells in the leukemia cell-derived HSP70-immunized mouse model.Methods: Balb/c mice and syngeneic A20 B-cell leukemia cell line were used in this study. HSP70 was purified from A20 cells or healthy mice liver tissue. After subcutaneous administration of A20-derived HSP70 (A20-HSP), liver-derived HSP70 (liver-HSP; control) to the healthy mice, the sera were harvested to perform following experiments. To detect anti-A20 antibodies, mean fluorescence intensity (MFI) of A20 cells with mice sera and FITC-conjugated anti-mouse-IgG was analyzed by flowcytometry. The sera were subjected to ELISA to detect the specific IgG against A20-HSP, or A20 secreted IgG (A20-Ig) as an A20-specific antigen. To investigate a contribution of A20-HSP70 specific CD4+ T-cell, expression of intracellular Th2-cytokine IL4 in the A20-HSP70 stimulated CD4+ T-cell in the HSP70-immunizaed mice was measured by flowcytometry. Complement dependent cytotoxicity (CDC) activities were determined by trypan blue uptake of mouse target cells (A20, YAC1: lymphoma, or T27A: myeloid leukemia) after incubation with mice sera and complement.Results: MIF of A20 with the sera from A20-HSP immunized mice (A20-HSP mice) was significantly higher than that from liver-HSP immunized mice (liver-HSP mice). IgG level against A20-HSP by ELISA was significantly increased in the A20-HSP mice compared with liver-HSP mice. The reactivities of A20-HSP mice sera against A20-HSP were completely lost by dissociation of the antigenic peptide from A20-HSP after ATP-treatment. Additionally, IgG level against A20-Ig in the A20-HSP mice was significantly higher than that in the liver-HSP mice, and this reactivity was blocked by preincubation of the sera with A20-idiotype derived peptide (A20-IP), which is the A20-specific peptide. A20-HSP70-reactive IL4-producing CD4 + T-cells in the A20-HSP mice are extremely more than those in the liver-HSP mice. The sera from A20-HSP mice showed no cytotoxic activity itself but showed significantly high CDC activity with complement against A20 but not to YAC-1 or T27A in vitro.Conclusions: Immunization with leukemia cell-derived HSP70 induces the leukemia-specific antibodies against peptides binding to leukemia cell-derived HSP70, including B-cell leukemia idiotypic peptide, via activation of the leukemia-specific CD4+ T-cell. In addition, leukemia-specific antibody-mediated CDC contributes to the eradication of leukemia cells. To utilize the leukemia-specific CDC activities induced by HSP-based immunotherapy would be a novel therapeutic strategy to eradicate leukemia cells in the patients with leukemia.

Augmented Antitumor Activity against B-Cell Lymphoma by a Combination of Monoclonal Antibodies Targeting TRAIL-R1 and CD20

Mapatumumab and lexatumumab are fully humanized, high-affinity immunoglobulin G(1 lambda) monoclonal antibodies (mAb) that target/activate the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2), respectively, triggering the extrinsic apoptotic pathway. Theoretically, synergistic antitumor activity should be observed by combining TRAIL-R mAbs with agents (e.g., rituximab) that activate the intrinsic apoptotic pathway. To this end, targeted antigen expression in a NHL-cell panel was evaluated by flow cytometry. NHL cells were exposed to mapatumumab or lexatumumab followed by rituximab, isotype, or RPMI. DNA synthesis was quantified by [(3)H]-thymidine incorporation assays. Induction of apoptosis was detected by flow-cytometric analysis. For antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) studies, standardized (51)Cr-release assays were done. We inoculated severe combined immunodeficiency (SCID) mouse with Raji cells i.v. The animals then were treated with various combinations of rituximab, mapatumumab, lexatumumab, and isotype alone or in combination. In vitro exposure to mapatumumab resulted in significant apoptosis (30-50%) and decreased DNA synthesis in sensitive lymphoma cells. Mapatumumab/rituximab combination resulted in a significant inhibition of cell proliferation (90% reduction) when compared with mapatumumab (60% reduction) or rituximab (5% reduction). In vivo, the median survival time of animals treated with mapatumumab and rituximab was longer (not reached) than those treated with rituximab monotherapy [33 days (95% confidence interval, 29-37), log-rank test, P = 0.05]. Mapatumumab induces apoptosis, cell growth arrest, ADCC, and CMC. The combination of mapatumumab plus rituximab is more effective in controlling lymphoma growth in vivo than either antibody. Rituximab and mapatumumab warrant further evaluation against B-cell lymphoma.

Modulation of Natural Killer Cell Activity, Antibody-Dependent Cellular Cytotoxicity, and Antibody-Dependent Complement-Mediated Cytotoxicity by Andrographolide in Normal and Ehrlich Ascites Carcinoma-Bearing Mice

Modulation of immune responses is highly relevant in tumor cell destruction. The present study is focused on the effect of Andrographis paniculata extract (APE) and its isolated compound andrographolide (ANDLE) on cell-mediated immune responses in normal and tumor-bearing control animals. Treatment with APE and ANDLE significantly enhanced natural killer cell activity in normal (APE, 46.82% cell lysis; ANDLE, 40.79% cell lysis) and tumor-bearing animals (APE, 48.66% cell lysis; ANDLE, 42.19% cell lysis) on the fifth day, and it was observed earlier than in tumor-bearing control animals (12.89% cell lysis on day 9). Antibody-dependent cellular cytotoxicity was also increased in APE (45.17% cell lysis on day 11) as well as ANDLE (39.92% cell lysis on day 11)-treated normal and tumor-bearing animals (APE, 47.39% cell lysis; ANDLE, 41.48% cell lysis on day 11) compared to untreated tumor-bearing control animals (maximum of 11.76% cell lysis on day 17). An early enhancement of antibody-dependent complement-mediated cytotoxicity was also observed by the administration of APE and ANDLE in normal as well as tumor-bearing animals. APE and ANDLE administration could significantly enhance the mitogen-induced proliferation of splenocyte, thymocyte, and bone marrow cells. Moreover, treatment of APE and ANDLE significantly elevated the production of interleukin-2 and interferon-gamma in normal and Ehrlich ascites carcinoma-bearing animals.

Effect of Biophytum sensitivum on Cell-Mediated Immune Response in Mice

Effect of Biophytum sensitivum on cell-mediated immune response was studied in normal as well as Ehrlich ascites tumor bearing BALB/c mice. Administration of Biophytum sensitivum significantly enhanced the proliferation of splenocytes, thymocytes and bone marrow cells by stimulating the mitogenic potential of various mitogens such as Lipopolysaccharide (LPS), Concanavalin A (Con A), Phytohaemagglutinin (PHA) and Poke Weed Mitogen (PWM). Natural killer (NK) cell activity was enhanced significantly by Biophytum sensitivum in both the normal (43.6% cell lysis on day 5) and the tumor bearing group (48.2% cell lysis on day 5), and it was found to be earlier than tumor bearing control animals (maximum of 13.4% cell lysis on day 9). Antibody dependent cellular cytotoxicity (ADCC) was also enhanced significantly in both Biophytum treated normal (35% cell lysis on day 7) as well as tumor bearing animals (40.2% cell lysis on day 7) compared to untreated control tumor bearing animals (maximum of 12.3% cell lysis on day 11). An early antibody dependent complement mediated cytotoxicity (ACC) was also observed in the Biophytum treated normal (22.6% cell lysis, on day 15) and tumor bearing animals (26.4% cell lysis, on day 15). Results of our present study suggest the immunomodulatory property of Biophytum sensitivum.

Effect of Punarnavine, an Alkaloid from Boerhaavia diffusa, on Cell-Mediated Immune Responses and TIMP-1 in B16F-10 Metastatic Melanoma-Bearing Mice

Effect of Punarnavine on the cell-mediated immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of Punarnavine enhanced Natural Killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared to the metastatic tumor-bearing control. Production of cytokines such as IL-2 and IFN-γ were significantly enhanced by the administration of Punarnavine compared to the untreated metastatic tumor-bearing control. Peaks of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α were significantly lowered by Punarnavine administration compared to metastatic control. The level and expression of TIMP-1 was also enhanced by the administration of Punarnavine compared to metastatic tumor bearing control. These results indicate Punarnavine could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.

Modulation of Cell-Mediated Immune Response in B16F-10 Melanoma-Induced Metastatic Tumor-Bearing C57BL/6 Mice by Sulforaphane

Effect of sulforaphane on cell-mediated immune response (CMI) was studied in B16F-10 melanoma-induced metastasis-bearing C57BL/6 mice. Administration of sulforaphane significantly enhanced natural killer (NK) cell activity in metastatic tumor-bearing animals (43.17% cell lysis, on day 5) and the activity was observed earlier than in tumor-bearing control animals (maximum of 9.76% cell lysis, on day 9). Antibody-dependent cellular cytotoxicity also was enhanced significantly in metastatic tumor-bearing animals (41.20% cell lysis on day 9) after sulforaphane administration compared with untreated control tumor-bearing animals (maximum of 12.62% cell lysis on day 15). An early antibody-dependent complement-mediated cytotoxicity also was observed in sulforaphane-treated tumor-bearing animals (26% cell lysis, on day 15). Administration of sulforaphane significantly enhanced the production of IL-2 and IFN-γ in metastatic tumor-bearing animals. In addition, sulforaphane significantly downregulated the serum levels of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and GM-CSF during metastasis. These data clearly suggest that sulforaphane effectively inhibited the spread of metastatic tumor cells through the stimulation of CMI, upregulation of IL-2 and IFN-γ, and downregulation of proinflammatory cytokines IL-1β, IL-6, TNF-α, and GM-CSF.