Enhanced antitumor activity of a combination treatment with a mouse/human chimeric anti-MK-1 antibody and lymphokine-activated killer cells in vitro and in a severe combined immunodeficient mouse xenograft model

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers. In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 V_H and Vκ genes to the human Cγ1 and Cκ genes, respectively. In the present study, we tested combination immunotherapy of Ch FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID) bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1 was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1 may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors. Received: 27 November 1998 / Accepted: 23 February 1999     

Theoretical studies on pyrimidine substituent derivatives as dual inhibitors of AP-1 and NF-κB

Theoretical studies on the three-dimensional (3D) quantitative structure-activity relationship (QSAR) and mechanisms of action of a series of pyrimidine substituent derivatives as dual inhibitors of AP-1 and NF-κB were carried out using comparative molecular field analysis (CoMFA) and docking methods. The established 3D-QSAR model exhibits a satisfying statistical quality and prediction ability. Docking results show somewhat lower average values of the flexible and rigid energy scores in the chosen binding sites. The docking analysis offers appropriate orientations and conformations of these compounds at the binding sites to both AP-1 and NF-κB in good agreement with the 3D-QSAR model from CoMFA. The combined CoMFA and docking study suggests the following substituent selections: substituent R₂ should be a kind of H–N–thienyl or CH₃–N–thienyl group; substituent R₅ should be a kind of COO–tBu or COOEt group; and substituent R₄ should be a CH₂CH₃ or 2-thienyl group. The docking analysis also shows that the binding sites fall just at the joint regions between AP-1 (or NF-κB) and DNA, where these compounds can effectively prevent free AP-1 and NF-κB from binding to DNA, and this may be the reason that derivatives with pyrimidine substituents have an inhibition function. In addition, a very interesting finding was that the binding sites of both AP-1 and NF-κB have a common structural characteristic, thereby providing a reasonable explanation for the dual inhibition functions of these compounds towards both AP-1 and NF-κB. These theoretical results help to deepen our understanding of the inhibition mechanism of these pyrimidine substituent derivatives, and will aid in directing further drug-molecular design.     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-κB

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-α, IL-1β, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-κB as indicated by inhibition of degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding, and NF-κB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.     

Human monocytes inhibit lymphokine-activated killer cell expansion in vitro

Depleting monocytes from human peripheral blood mononuclear cells (PBMC) enhances the in vitro activation of lymphokine-activated killer (LAK) cells. To determine if monocytes also altered LAK-cell expansion, we evaluated two methods of depleting monocytes from PBMC: nylon wool adherence (NWA) and phenylalanine methyl ester (PME) treatment. Both methods of depleting monocytes enhanced interleukin-2 (IL-2) driven, LAK-cell expansion; LAK expansion, however, was significantly greater after depletion with NWA than after PME. LAK cytotoxicity after NWA and PME depletion was equivalent. The degree of monocyte depletion, determined by evaluating morphology and the number of Leu-M3 (CD14) positive cells, and the proliferation of Leu 19 (CD56), OKT-3 (CD3), Leu2 (CD8), and Leu 3a (CD4) positive cells was also equivalent. Exposure of IL-2 activated cells to PME did not alter their cytotoxic activity. However, sequential treatment of PBMC with NWA, then PME, or with PME and then NWA, resulted in reduced expansion. This reduction in expansion was similar to PBMC treated with PME alone. Exposure of PME-depleted cells to nylon wool or to supernatants obtained from cells adherent to nylon wool further decreased LAK expansion relative to cells treated with NWA alone. We conclude that even at relatively low cell density, human monocytes markedly inhibit LAK-cell expansion in IL-2 driven PBMC cultures. Further, depletion of monocytes by NWA adherence is more effective than by treatment with PME, possibly due to subtle cellular damage induced by this latter treatment. These findings have implication for the in vitro and in vivo generation of LAK-cells by IL-2.     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)