ALBERT

Description: Tru 16.4 is a single chain protein with a modified CD37 binding Fv domain linked to a modified human IgG1 hinge, CH2 and CH3 domains. It is a member of a novel composition class called small modular immunopharmaceuticals (SMIP) construct. CD37 is a stable marker for B-cell chronic lymphocytic leukemia (BCLL) and non-Hodgkins lymphoma (NHL). Although radiolabeled murine anti-CD37 antibody MB-1 has been evaluated in NHL patients and shown to be able to target tumors, efficacy was not established. There has been no evaluation of an unconjugated anti-CD37 antibody in human clinical trials. Here we report that Tru 16.4, binds to CD37 on primary CLL cells surface, and induces caspase-independent apoptosis that is further enhanced by a secondary crosslinking with goat anti-human antibody. The percentage of dead cell population over control based upon Annexin V/PI staining of 12 BCLL patient samples (28.3%, SD 15.4%) is significantly greater than that of isotype control, trastuzumab (3.4%, SD 7.6%). The expression of CD37 on T cells is low, and the exposure of Tru 16.4 under the same conditions does not demonstrate significant binding or induce noticeable apoptosis on normal T cells at 24 hours (0.47%, SD 0.99%). This provides a selective advantage for BCLL antibody therapy. Results from a comparison study utilizing 6 additional patient samples demonstrated that Tru 16.4 induced apoptosis (31.8%, SD 12.2%) is significantly higher than that of the anti-CD20 antibody, rituximab (21.8%, SD 5.25%) and similar to that observed with the anti-CD52 antibody, alemtuzumab (36.6%, SD 19.5%). The cell death induced by Tru 16.4 is dependent upon dose and duration of treatment, indicating 5μg/mL as a saturation concentration and 24 hours as the optimal time for maximal in vitro apoptosis. The degree of CLL cell death was found to be directly proportionate to cell surface membrane expression of CD37, based on nonparametric Spearman rank correlation analysis of the mean fluoresecnce intensity (MFI) after PE-labelled anti-CD37 antibody immunostaining and percentages of Annexin V/PI positive cells, with a correlation factor rs = 0.80 ( P 〈 0.01). Ongoing studies suggest Tru 16.4 induces intracellular signaling following CD37 antigen ligation and current efforts are focused on identifying the specific death pathways mediated by this treatment. Overall, these findings indicate that Tru 16.4 mediates B-cell specific apoptosis through a novel caspase independent mechanism and supports effort for further clinical investigation in CLL.

Description: Induction of chemokine gene expression from peripheral blood mononuclear cells (PBMCs) stimulated by proinflammatory cytokines plays an important role in both wound repair and response to infectious agents. In the present study, we show that the proinflammatory cytokine interleukin-6 (IL-6) potently induced mRNA expression and secretion of the CC chemokine monocyte chemotactic protein 1 (MCP-1) in PBMCs. In addition, because human immunodeficiency virus (HIV) infection in vivo and in vitro has been shown to dysregulate the production of and/or the response to cytokines, PBMCs from both healthy uninfected and HIV-infected individuals were studied for their constitutive and IL-6–induced expression of MCP-1. No substantial differences were observed between the two groups of individuals. In addition, IL-6 upregulated MCP-1 expression in the promonocytic cell line U937 and in its chronically HIV-infected counterpart, U1. In these cell lines, IL-6 selectively induced MCP-1 and not other chemokines, including regulated upon activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and IL-8. IL-6 induction of MCP-1 was partially inhibited by hydrocortisone in U1 cells. Thus, IL-6 activates PBMCs to secrete MCP-1, a CC chemokine pivotal for monocyte recruitment in tissue and organs in which important inflammatory events occur.

Description: Uric acid, a final metabolite of purine metabolism has gained attention for its pathogenic role in the development of hypertension, vascular disease, renal disease, and cardiovascular events. Recent studies have shown that soluble uric acid induces proliferation as well as synthesis of proinflammatory chemokine, monocyte chemoattractant protein 1 in rat vascular smooth muscle cells. It also stimulates human monolayer cells to produce IL-1B. IL-6 and TNF alpha. However, high mobility group box 1(HMGB1), a non histone DNA binding protein which is recently identified as a proinflammatory cytokine and can be released from cells of the macrophage/monocyte lineage by proinflammatory stimuli or by cells undergoing necrotic cell death pathway. In the present study, we hypothesized that soluble uric acid may stimulates monocytes/macrophages to release HMGB1. Here we demonstrate that crystal and endotoxin free uric acid triggers the release of HMGB1 in a time and dose dependant fashion in mouse macrophage cells, Raw 264.7, human leukemic promonocytes(THP-1 cells) as well as in macrophages but not in fibroblast obtained from synovial fluid of rheumatoid arthritis patients. Moreover, translocation of HMGB1 to the cytoplasm before secreted in the extracellular milieu was significantly detected within 6h of Uric acid exposure. No loss of cell viability was observed in our experimental condition as judged by trypan blue exclusion, MTT reduction and LDH release assays, indicating that HMGB1 release was not due to cell death. Uric acid treatment resulted in the activation of p38 mitogen activated protein kinase(MAPK), c-Jun N terminal kinase(JNK) and extracellular regulatroy kinase (ERK1/2). Inhibition of each of p38 MAPK, ERK1/2 and JNK by using potent and selective inhibitors, SB203580, U0126 and SP60012 respectively, almost completely blocked uric acid-induced HMGB1 release. Furthermore, Curcumin, a potent inhibitor of the transcription factor, activator protein 1(AP-1) strongly suppressed HMGB1 release, indicating that these signaling patways are essential for the induction of HMGB1 release in response to uric acid. In conclusion, our data suggest that uric acid may regulate critical proinflammatory pathways at least in part through its action as a monocyte/macrophage derived potent proinflammatory cytokine, HMGB1 release. This may play pathologic role for hyperuricemia-induced morbidities including arthritis, atherosclerosis, renal disease and so on.

Description: High Mobility Group box 1(HMGB1) is an abundant DNA-binding protein that acts as a proinflammatory cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Moreover, an increased HMGB1 in the circulation of septic patients may induce multi-organ failure and lethality. However, very recent observations suggest that the protein also acts as an innate adjuvant, stem cell chemoattractant and growth factor. Thus only systemic and circulatory HMGB1 may induce morbidity and mortality, however, localized HMGB1 may have beneficial effects. Therefore, we serially examined the serum HMGB1 level in patients with various diseases, and also evaluated the significance of the protein. We demonstrate here how HMGB1 is localized and acts as an immune-adjuvant and a repairing factor in damaged tissue. We first established specific ELISA method to measure HMGB1. An increased level of HMGB1 was detected in the serum from patients with sever sepsis, infections, malignancy and so on. However, serum HMGB1 concentrations were fluctuated during the clinical course, and could not be concluded as a lethal mediator as previously reported. Next we investigated the reason of dynamic fluctuations of the protein in the circulation. Based on our findings, we proposed that this fluctuation of HMGB1 concentrations may be mediated by at least following three fashions; 1) proteolytic degradation by plasmin and thrombin, 2) endothelial thrombomodulin(TM) adsorption, and 3) generation of antibody against the protein. We observed that plasmin efficiently degraded HMGB1 into small fragments. However, interestingly the generated fragments of the protein still possess an ability to produce TNFa in macrophages through an undefined pathway. TM binds the protein on its N-terminus lectin-like domain. Binding of HMGB1 to TM resulted in decrement of TM’s cofactor activity to activate protein C by thrombin. HMGB1 bound to TM was gradually degraded by thrombin. These may be a system to localize HMGB1 only in injury sites where TM is down-regulated or disappeared through endothelial-loss. This may exert endothelial defense system against extracellular HMGB1 in severe tissue injury. Another possibility is that the generated antibody against HMGB1 may neutralize the proinflammatory action of the protein. In this context, we found that some of the antibodies against HMGB1 have the characteristics of P-ANCA(perinuclear anti-neutrophil cytoplasmic antibody). This may alter the phenotype of the underlining diseases. In conclusion, we suggest that HMGB1 is not merely a lethal mediator, but a kind of “testament” mediator of cell necrosis or invasive attacks to dendritic cells.

Description: We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3’-azido-3’-deoxythimidine (AZT) or cell-exposure to heat-inactivated (▵°) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain.

Description: Hu1D10 (apolizumab), a humanized HLA-DR beta-chain-specific antibody directed to the 1D10 antigen, has been shown to be cytotoxic towards primary B-cell chronic lymphocytic leukemia (CLL) cells and is currently in clinical trials for this disease. Other second generation HLA-DR antibodies are also in late pre-clinical development at this time. We previously reported that in vitro Hu1D10 treatment of CLL cells resulted in generation of reactive oxygen species (ROS); however, antioxidant treatment did not modulate the cytotoxicity observed. It is known that CLL cells have a compromised antioxidant defense system, as evident from the low activities of the major antioxidant enzymes superoxide dismutase and catalase, and the accumulation of degradation products like malonaldialdehyde and 8-oxo-deoxyguanosine. Therefore, we hypothesized that CLL cells would be susceptible to damage by ROS generating agents, and that ROS generating agents would enhance the cytotoxicity of Hu1D10. Furthermore, CLL cells are reported to have high concentrations of the antioxidant glutathione (GSH), thus we expect GSH depletion by buthionine sulfoximine (BSO) treatment to enhance the cytotoxicity of Hu1D10. Arsenic trioxide (ATO), an agent that is approved for treatment of relapsed acute promyelocytic leukemia, is believed to exert its cytotoxicity through the generation of ROS and thiol depletion. The efficacy of ATO is enhanced by ascorbic acid. At high concentrations, ascorbic acid is also capable of generating ROS . We found that ATO was cytotoxic to both CLL cells and the 697 B-cell line in a dose dependant manner. Ascorbic acid alone was cytotoxic to CLL cells and 697 cells at very high doses, but not at 1mM. As a sole agent, ATO at clinically relevant concentrations (0.5 – 2 μM) was not cytotoxic towards 697 or CLL cells at 24 hrs; however, ATO (1 μM) in combination with 1mM ascorbic acid was toxic toward 697 cells as measured by annexin V-FITC/propidium iodide staining with FACS analysis (viability in Control = 94.5% ± 1.1, ATO + ascorbate = 31.7% ± 0.8). We found a similar cytotoxic response in two primary CLL cell samples treated with ATO (1 μM) + ascorbic acid (1 mM) [CLL-1 viability in Control = 64.4% ± 0.3, ATO + ascorbate = 12.3% ± 0.2; CLL-2 viability in Control = 83.3 ± 0.2, ATO + ascorbate = 30.9% ± 0.3]. Depletion of glutathione by BSO enhanced ATO/ascorbate cytotoxicity in 697 cells by 40%. However, this treatment was less effective in enhancing the cytotoxicity of Hu1D10 in 697 cells (8.7% increase). We next investigated whether ATO/ascorbate co-administration would be able to enhance the cytotoxicity of Hu1D10 as hypothesized. The addition of ATO (1 μM) and ascorbic acid (1 mM) enhanced the cytotoxicity of Hu1D10 towards 697 cells and CLL cells (viability in 3 CLL patients are: Control = 73.2% ± 10.5, Hu1D10 = 43.8% ± 16, Hu1D10 + ATO/ascorbate = 20.2% ±11). Combination treatment of CLL cells or 697 cells with ATO and ascorbic acid is cytotoxic at clinically relevant concentrations. Furthermore, the toxicity of Hu1D10 toward CLL and 697 cells is increased by the addition of ATO and ascorbic acid. These results suggest that ATO and ascorbic acid alone, or in combination with the antibody Hu1D10 could emerge as effective therapies for the treatment of CLL.

Description: Previous studies have demonstrated that both normal B-cells and CLL cells are susceptible to redox stress. The expanded porphyrin, motexafin gadolinium (MGd), is a tumor selective redox active drug that reacts with various intracellular reducing metabolites and protein thiols to generate reactive oxygen species (ROS). Multiple trials in solid tumors have established that this agent is well tolerated and not associated with myelosuppression, although its toxicity profile has not been explored in hematologic malignancies. We therefore initiated a pilot phase I trial of MGd in previously treated CLL and small lymphocytic lymphoma (SLL) to assess the toxicity, pharmacokinetics, and preliminary clinical activity of MGd and to perform pharmacodynamic studies to confirm ROS generation in vivo. MGd was administered 5mg/kg/day IV for 5 days every three weeks until disease progression. MGd uptake was measured in CLL cells by flow cytometry. Effects of MGd treatment on Protein Kinase B (AKT) phosphorylation in vivo were examined, as we have previously demonstrated that this kinase is activated by ROS in CLL cells. Thirteen patients (pts) were enrolled with a median age of 66 years (range 54–80) and a median of 4 prior therapies (range 2–9); 12 pts had fludarabine-refractory disease. Median WBC was 26.9 (range 5.4–152.6); median platelet count was 95,000 (range 33,000–214.000) with 7 pts 〈 100,000 and 3 pts 〈 50,000. The most common Grade 1–2 adverse events were transient skin discoloration (13), asthenia (6), bone pain (2), and diarrhea (5). Five pts had possibly drug related Grade 3 toxicity, including neutropenia (2), anemia (2), hypophosphatemia (2), hypocalcemia (1) and cellulitis (1). No pts suffered treatment delay due to toxicity. Evidence of tumor activity was seen in three pts and included decrease in WBC, nodes and/or splenomegaly, although no pts met NCI 96 response criteria. One responding pt during cycle 2 of therapy developed a bowel perforation and was found to have massive tumor necrosis of large cell transformation at the perforation site at surgery. MGd uptake into CLL cells was analyzed 24 hours after dose 1 and 4 in 9 pts. Median fluorescence intensity (MFI) increased 1.1 to 1.4 fold (median 1.2 ± 0.1) over background after dose 1 and 1.5 to 4.0 fold (median 2.2 ± 0.8) after dose 4. AKT phosphorylation increased in serial samples obtained during treatment in a subset of patients, supporting the biologic activity of this agent in vivo. In conclusion, these data suggest that MGd has modest clinical activity in relapsed and refractory CLL when given as a single agent. Drug uptake into tumor cells was confirmed, and evidence of biologic activity was demonstrated by in vivo response and phosphorylation of AKT in a subset of patients. Pharmacodynamic studies suggest that alternative schedules of administration may be used to improve tumor response. Based upon these data, follow-up trials using optimized dosing regimens of MGd are in progress in less heavily treated CLL patients.

Description: CD4, the primary receptor for entry of HIV, is known to be expressed on T cells and monocytes/macrophages; healthy natural killer (NK) lymphocytes; in vitro human herpesvirus 6 (HHV6)–infected CD8+, NK, and γδ T lymphocytes; CD34+ progenitor cells; and a subset of eosinophils and basophils. We here report the unconventional expression of CD4 at the surface of peripheral blood neutrophils derived from 4 of 51 (7.8%) HIV-1–infected and 3 of 25 (12%) uninfected donors, with similar frequency within the 2 groups. The percentage of CD4+ neutrophils ranged from 39% to 97% of the total neutrophil population. Both surface and cytoplasmic forms of CD4 were present in neutrophils. Quantitative RNA polymerase chain reaction (PCR) revealed that neutrophils contain levels of CD4 mRNA comparable to those of peripheral blood mononuclear cells derived from the same donor. The conformation of CD4 expressed at the surface of neutrophils was similar to that of CD4 expressed on T lymphocytes as determined by the binding of monoclonal antibodies specific for conformational epitopes and the binding of recombinant HIV-1 gp120. Thus, our data provide evidence that neutrophils express endogenous CD4 and bind HIV. Owing to their abundance in peripheral blood, CD4+ neutrophils may influence significantly the biodistribution of HIV delivering it to sites of inflammation or to additional tissue reservoirs.

Description: Induction of chemokine gene expression from peripheral blood mononuclear cells (PBMCs) stimulated by proinflammatory cytokines plays an important role in both wound repair and response to infectious agents. In the present study, we show that the proinflammatory cytokine interleukin-6 (IL-6) potently induced mRNA expression and secretion of the CC chemokine monocyte chemotactic protein 1 (MCP-1) in PBMCs. In addition, because human immunodeficiency virus (HIV) infection in vivo and in vitro has been shown to dysregulate the production of and/or the response to cytokines, PBMCs from both healthy uninfected and HIV-infected individuals were studied for their constitutive and IL-6–induced expression of MCP-1. No substantial differences were observed between the two groups of individuals. In addition, IL-6 upregulated MCP-1 expression in the promonocytic cell line U937 and in its chronically HIV-infected counterpart, U1. In these cell lines, IL-6 selectively induced MCP-1 and not other chemokines, including regulated upon activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and IL-8. IL-6 induction of MCP-1 was partially inhibited by hydrocortisone in U1 cells. Thus, IL-6 activates PBMCs to secrete MCP-1, a CC chemokine pivotal for monocyte recruitment in tissue and organs in which important inflammatory events occur.

Description: We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Abl–positive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys Bcr-Abl–positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl–negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210Bcr-Abl fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl–positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl–negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of Bcr-Abl kinase leading to activation of p38 mitogen-activated protein (MAP) kinase may play an important role in the anti-CML activity of Chl.