Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

Theremarkableabilityofhumantumornecrosisfactor(hTNF-a)istokillmanymalignantcelllinesinvitroorinvivoselectivelyandhavealmostnotoxicityfornormaltissuecells[1,2].However,manysideeffectsofhTNF-ainclinictrialshaveseverelylimiteditsapplicationincancertreatment[3].Recently,alotofworkhasbeendoneforimprovinghTNF-abymeansofproteinengineeringtoobtainnovelhTNF-amutantswithhighcytotoxcityandreducedsystematictoxicity.Yamagishietal.pointedoutthattheessentialfourregionsformaintainingtheactivityofhTNF-aw…     

Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-α using computer modeling and deletion mutant technique

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-α were cloned, and according to the translated sequence of amino acids, the spatial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-α was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-α. hTNF-α molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92–157 of hTNF-α, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF-α 141–146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-α were specially recognized by Z12 antibody.     

Expression of a functional human tumor necrosis factor-α (hTNF-α) in yeast<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The recombinant soluble human tumor necrosis factor-alpha (hTNF-α) was expressed in a yeastSaccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-α was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybridADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-α existed among transformants, the higher expression was obtained with theGPD promoter. Expressed hTNF-α protein (rhTNF-α) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-α, indicated that the secreted rhTNF-α was bioactive and its doseresponse was improved eight to ten times over that of theE. coli-derived rhTNF-α.     

Establishment of a human fibroblast cell line producing tumor necrosis factor α (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture

Summary To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor α (hTNF-α) has been developed by introducing hTNF-α cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-α antibody, its inhibitory effects were more marked than the purified human natural TNF-α itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-α. However, interferons α, β, and γ were undetectable in the KMST-6/TNF CM.     

Determinants of miniprotein stability: can anything replace a buried H-bonded Trp sidechain?

Summary A novel 20-residue fold, designated the ‘Trp-cage’ motif, has been shown to be 98+% folded in both water and 30 vol-% trifluoroethanol solution. Folding is cooperative and hydrophobically driven, resulting in the burial of the Trp sidechain and a stable H-bond from the Trp-εNH to a sequence remote backbone carbonyl. In the present study the effects of replacing the Trp with His, Phe and both isomers of β-naphthylalanine are examined. The results suggest that the hydrophobic cluster is a specific interaction of proline rings with the indole ring which can be partially mimicked by a naphthalene ring. The His and Phe mutants are completely unfolded in aqueous medium. The naphthylalanine mutants form a stable hydrophobic cluster in 30% trifluoroethanol, but are less stable in water than the native structure.     

Mutational analysis on the function of the SWAP-70 PH domain

Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant cancers worldwide, especially in Taiwan. Estrogen receptors (ERs) have been reported to play either a proliferation- or apoptosis-enhancing role in the differentiation of cancers, including HCC. In a previous experiment, we showed that transient overexpressed estrogen receptor-α induced early stage HCC cell line Hep 3B cell apoptosis by increasing the hTNF-α gene expression in a ligand-independent manner. To further clarify if the apoptotic effect occurs in poorly differentiated HCC cell line, HA22T, and elucidate the roles of ERs and TNF-α, DNA fragmentation and caspase activity were measured in late stage HCC cell line, HA22T, by measuring the expression of hER-α and hER-β using a Tetracycline-induciable system (Tet-on). Increased DNA fragmentation and caspase-3 activity were found in hERβ-overexpressed HA22T cells treated with estrogen (10⁻⁸ M) but not in hERα-overexpressed HA22T cells. Using RT-PCR/PCR and western blotting in HA22T cells, overexpressed hER-β was also found to increase the expression of hTNF-α mRNA and induce hTNF-α-dependent luciferase activity in a ligand-dependent manner. Additionally, LPS treatment and hER-β overexpression both enhance caspase-8 activities, whereas neither hER-β nor E₂ treatment affected caspase-9 activities. In addition, the overexpressed hER-β plus E₂ enhanced DNA fragmentation and caspase-8 activities were only partially reduced by anti-hTNF-α (0.1ng/ml), which was possibly due to the involvement of P53 and TGF-β. Taken together, our data indicates that overexpressed hER-β but not hER-α may induce caspase-8-mediated apoptosis by increasing the hTNF-α gene expression in a ligand-dependent manner in poorly differentiated HA22T cells. (Mol Cell Biochem xxx: 1–9, 2005)Shares equally contribution Contract grant sponsor: National Science Council; Contract grant number: NSC 91-2314-B-075A-006, NSC 92-2314-B-075A-014.     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2)

Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 μM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS -subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially -methyltryptophan (MT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on MT, indicating that it might be possible to use ASA2 as a selectable marker gene and MT as the selection agent.     

Amino Acids that Confer Transport of Raffinose and Maltose Sugars in the Raffinose Permease (RafB) of Escherichia coli as Implicated by Spontaneous Mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391

In order to identify amino acid residues in the Escherichia coli raffinose-H⁺ permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and α-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35→Ala (MT1); one mutant had Ile-391→Ser (MT2); and two mutants had Ser-138→Asp, Ser-139→Leu and Gly-389→Ala (MT3). Transport studies of [³H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P ≤ 0.05) than cells harboring wild-type RafB. However, [¹⁴C]-raffinose uptake was reduced in all mutant cells (P ≤ 0.05) with MT1, MT2 and MT3 mutants compared to cells harboring wild-type RafB. Kinetic analysis showed enhanced apparent K _m values for maltose and reduced V _max/ K _m ratios for raffinose compared to wild-type values. The apparent K _i value of maltose for RafB indicates a competitive relationship between maltose and raffinose. Maltose “uphill” accumulation was greater for mutants (P ≤ 0.05) than for cells with wild-type RafB. Thus, we implicate residues in RafB that are responsible for raffinose transport and suggest that the substituted residues in RafB dictate structures that enhance transport of maltose.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

<Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid coupled human recombinant TNFα exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor α (TNFα) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFα. NeuAc-coupled TNFα (NeuAc-TNFα) exhibited reduced activities in vitro by about threefold compared to native TNFα. In this study, we examined a variety of TNFα activities in vivo. NeuAc-TNFα reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFα in the down-regulation of the serum level of glucose. However, NeuAc-TNFα was more potent than TNFα in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFα was less toxic to mice. In addition, NeuAc-TNFα exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFα with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.     

Prediction of mutant activity and its application in molecular design of tumor necrosis factor-a

Two models for prediction of the activity and stability of site-directed mutagenesis on tumor necrosis factor-α are established. The models are based on straightforward structural considerations, which do not require the elaboration of sitedirected mutagenesis on the protein core and the hydrophobic surface area by analyzing the pmperties of the mutated amino acid residues. The reliabilities of the models have been tested by analyzing the mutants of tumor necrosis factor-α (TNF-α) whose two leucine residues (L29, L157) were mutated. Based on these models, a TNFα mutant with high activity was created by molecular design. Project supported by the Chinese National High Technology Development Program.     

Structure-activity relationships of recombinant human interleukin 2

Structure-activity relationships of recombinant human interleukin 2 were investigated by preparation, purification, and characterization of 21 missense mutants. A key role for residue Phe42 in the high-affinity interaction with receptor was indicated by (a) the reduction of 5-10-fold in binding affinity and bioactivity upon mutation of this residue to Ala and (b) the lack of evidence for conformational perturbation in Phe42----Ala in comparison with the wild-type protein as investigated by intrinsic fluorescence, second-derivative UV spectroscopy, electrophoresis, and reversed-phase HPLC, suggesting that the drop in binding is a direct effect of removal of the aromatic ring. In contrast, the conservative mutations Phe42----Tyr and Phe42----Trp did not cause significant reductions in bioactivity. UV and fluorescence spectra indicated approximately 60% overall exposure to solvent of tyrosines in the wild-type molecule, the tryptophan (residue 121) being buried; fluorescence data also showed that Trp42 in Phe42----Trp is likely to be within 1 nm of Trp121 and about 50% exposed to solvent. Phe44----Ala, Cys105----Ala, and Trp121----Tyr also exhibited reduced bioactivity, but these mutants are conformationally perturbed relative to wild type. None of the remaining mutants had detectably reduced bioactivity, even though several showed signs of altered conformation. Four mutants were recovered in very low yield, probably because of defective refolding.     

Metallothionein induction in freshly isolated rat hepatocytes

The control of metallothionein (MT) synthesis was investigated in freshly prepared rat hepatocytes in experiments of short-term duration. Viability and metabolic function were maintained in incubations of 6-h duration. MT synthesis was measurable in hepatocytes from fed rats at Zn concentrations down to 1 μM. Zn and dexamethasone induced concentration-dependent increases in the synthesis of MT with maximal increases above the 5-h control of 3.2- and 2.5-fold, respectively. Zn induction of MT was first measurable at 2 h and was inhibited by actinomycin C. Although initial (0 h) MT concentrations in hepatocytes from fasted rats were double those from fed rats, after 6-h incubation in the presence of 50 μM Zn, the fasted rat hepatocytes showed only half the MT concentrations of the fed rat hepatocytes. Glucagon and interleukin-6 (IL-6) were less effective inducers and increased MT synthesis by 28 and 17%, respectively. IL-6 (100 U/mL) was found to have an additive effect on MT synthesis above that of Zn alone (1–50 μM) or Zn plus dexamethasone (1 μM). A supernatant from LPS-stimulated macrophages increased MT synthesis by 40%. The basal MT synthesis was not increased by either tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1). All incubations were carried out in the presence of RPMI 1640 medium with Hepes (20 mM), bicarbonate (24 mM), and fatty acid-free albumin (FAFA; 0.5% w/v). MT synthesis was also seen using Krebs bicarbonate buffer with glucose (10 mM), Hepes (20 mM), and FAFA (0.5% w/v), and although the level of MT synthesis was less than in RPMI, the increases in concentrations of MT at 5 h were 225, 139, 36 and 20% for Zn, dexamethasone, glucagon, and control, respectively. It is concluded that MT synthesis occurs in freshly prepared hepatocytes and that these cells are responsive to some of the established inducers of MT. This system enables the study of MT synthesis in individual rats in various metabolic and pathological states.     

Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima

Some α-amylases besides catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch are also capable of carrying out some transglycosylation activity. The importance of aromatic residues near the catalytic site in determining the ratio of these two competing activities has been remarked in the past. In the present work we investigated the role of residue 260 in the product profile of the α-amylase AmyA from Thermotoga maritima. This phenylalanine residue, two positions after the glutamic acid/base catalyst was substituted by both tryptophan and glycine residues, showing opposite behaviors. The tryptophan mutant displayed a very similar product profile pattern to that of the wild-type enzyme; while the mutant Phe260Gly showed a higher transglycosylation/hydrolysis ratio. When the Phe260Trp mutation was constructed in the context of His222Gln, a mutant we have already reported with an increased transglycosylation/hydrolysis ratio and a higher alcoholysis activity, the resultant enzyme showed an apparent higher hydrolysis/transglycosylation ratio and a change to shorter products pattern than the single mutant enzyme, still maintaining the increased alcoholytic activity provided by the His222Gln mutation. The mutant Phe260Gly, on the other hand showed by itself a higher alcoholytic activity, similar to that of the His222Gln mutant.     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

A mutant induced in the malting barley cv Triumph with reduced dormancy and ABA response

 Induced mutants in the barley cultivar Triumph have been screened for reduced dormancy. One line, which germinated readily 2 weeks after harvest, was classified as ABA-insensitive, since it could tolerate a ten-fold increase in ABA, compared to its parent, before germination was inhibited. This mutant, designated TL43, was genotypically similar to Triumph and phenotypically similar under Scottish growing conditions, except for a slightly reduced grain size. In Spain, it showed considerable reductions in both grain yield and plant height, suggesting that it was less widely adapted than its parent. Levels of α-amylase activity were increased at both sites. The mutant appeared to be different from those with ABA insensitivity or altered dormancy previously documented in either barley or Arabidopsis. Received: 23 March 1998 / Accepted: 13 August 1998