Specific inhibition of the reaction between a tumor-inhibitory antibody and the ErbB-2 receptor by a mimotope derived from a phage display library

Overexpression of ErbB-2, a coreceptor for stroma-derived growth factors, is involved in malignancies of epithelial tissues, and a humanized antibody to ErbB-2 was shown to be therapeutic in a clinical setting. In an effort to understand and enhance immunotherapy, the laboratory has raised several tumor inhibitory monoclonal antibodies (mAb), including mAb L26 that blocks inter-receptor interactions. Here the application of the phage display methodology for the isolation of a phage clone that specifically recognizes mAb L26 is described. The isolated mimetic peptide (mimotope) specifically inhibited the binding of mAb L26 to ErbB-2 overexpressing cells. No sequence homology was found between the mimotope and ErbB-2. implying that it mimics a conformational structure of the receptor. Preliminary studies showed that the lead peptide can be truncated by removal of two to three amino acids from either the N- or C-terminal end without drastically affecting the inhibitory properties of the mimotope. A tryptophan'glycine residue at the C-terminus and a lysine at the N-terminus of the peptide seemed to play a role in its ability to compete with L26 antibody for binding to ErbB-2 overexpressing cells. These results highlight the potential of active immunization with conformation mimicking peptides in ErbB-2 overexpressing tumors.     

A mimotope defined by phage display inhibits IgE binding to the plant panallergen profilin

Birch pollen and mugwort pollen allergies are often associated with hypersensitivity to plant foods. This clinical and serological cross-reactivity is mediated by IgE antibodies reacting with homologous proteins in pollen and food. Cross-reacting homologs of the important birch pollen allergen Bet v 2 (profilin) could be detected in other pollen, fruits, nuts, and vegetables, such as celery tuber. We purified IgG/IgE antibodies from the serum of an exclusively profilin-allergic patient using affinity columns either coupled with protein extracts from mugwort pollen, birch pollen, or celery tuber. Constrained and unconstrained random nonapeptide libraries were pooled and screened with the anti-profilin antibody preparations to define cross-reactive ligands. Specific ligands were enriched by successive panning rounds using the profilin-specific antibodies in series. After the last panning round enriched phage clones were screened with purified profilin-specific antibodies and IgE-binding clones were sequenced. Five out of eight positive clones (62.5 %) displayed the same circular peptide CAISGGYPVC. This peptide was synthesized and examined for its ability to inhibit IgE binding to blotted mugwort pollen, birch pollen, or celery tuber profilin. Inhibition studies showed reduction of IgE binding to profilins in all three protein extracts. As the sequence of the mimotope did not show any homology to the known birch profilin sequence this peptide is considered to mimic a common conformational IgE epitope for these examined profilins.     

应用噬菌体随机肽库技术筛选SARS-CoV S蛋白模拟表位

目的筛选SARS病毒(Severe acutere spiratorysyndromeas sociated coronavirus,SARSCoV)结构蛋白S(Spike)特异性的模拟表位,为抗SARS疫苗研究提供基础。方法以抗SARS病毒S蛋白的单克隆抗体为固相筛选分子,对人工合成的噬菌体随机12肽库进行5轮“吸附洗脱扩增”的筛选,随机挑选30个克隆,经噬菌体酶联免疫吸附(ELISA)法和交叉反应实验鉴定阳性克隆,再进行DNA序列分析和竞争抑制结合实验,以确定SARS病毒S蛋白的模拟表位。结果经噬菌体富集后,从随机挑选的30个克隆中得到了29个编码8种12肽的阳性克隆,8条肽与S蛋白的320～350氨基酸序列有较高同源性,确定YF、W、E和K氨基酸残基为模拟表位的骨架结构。结论用噬菌体12肽库成功筛选到了SARS病毒S蛋白的模拟表位,为基于S蛋白的肽疫苗研制提供了基础。     

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope

One of the proposed approaches in cancer therapy is to induce and direct the patient’s own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.     

表达胃癌MG7-Ag模拟表位的减毒鼠伤寒杆菌疫苗的研制和初步鉴定

目的 研制可表达胃癌MG7－Ag模拟表位的减毒鼠伤寒杆菌疫苗。方法 将NcoI位点和MG7－Ag模拟表位编码序列的互补序列设计到上游引物的5′端。以含有HBcAg全序列的质粒p1.2Ⅱ为模板，通过PCR扩增，获得MG7－Ag模拟表位与HBcAg的融合基因。将目的基因插入载体pUCm-T，经酶切鉴定和序列测定证实后，再亚克隆至与减毒鼠伤寒杆菌X4550互补的质粒pYA3341中，再以此重组质粒转化减毒鼠伤寒杆菌X4550。结果 序列测定证实，PCR产物为胃癌MG7－Ag模拟表位与HBcAg的融合基因片段；最终得到含有目的基因片段的重组表达载体pYA3341。重组质粒在X4550中的表达产物，经Western blot表明，在相对分子质量（Mr)约在22000处有1条可与抗MG7 mAb特异性结合的多肽表位。结论 成功地研制可表达胃癌MG7－Ag模拟表位减毒鼠伤寒杆菌疫苗，为进一步研究其在胃癌免疫治疗中的作用奠定了基础。     

A mimotope peptide-based vaccine against Schistosoma mansoni: synthesis and characterization

A panel of four mimotopes of the epitope recognized by the highly protective monoclonal antibody against Schistosoma mansoni (152-66-9B) was obtained by screening a solid-phase 8mer random peptide library. Three of the four mimotopes (p28, p29 and p30) were efficiently recognized in an in vitro radioimmunoassay by the monoclonal antibody and by sera from infected mice and one (p30) induced in vitro proliferation of primed lymphocytes. When the mimotopes were conjugated to bovine serum albumin (BSA) and the conjugates used to immunize C57BL/6J mice, only the p30-BSA-induced antibodies which were effective at complement-mediated killing of schistosomula. The level of complement-mediated killing obtained with the anti-p30 antibodies was comparable to that seen with serum from mice immunized with the protective 9B-antigen. Furthermore, following challenge infection of mimotope-BSA-immunized mice, a greater than 40% reduction in worm burden was observed in p30-BSA-immunized mice, a level comparable to that seen following immunization with the intact 9B-antigen. These results show that a simple synthetic peptide immunogen comprising an eight-amino acid mimotope of a conformational epitope on the 9B-antigen can induce protective immune responses against S. mansoni that are comparable to those obtained following immunization with the far more complex intact antigen. This mimotope may well represent a potential component of a synthetic peptide vaccine against S. mansoni. The inclusion of other B-cell- and T-cell-stimulating synthetic epitopes in such a vaccine, together with a more appropriate carrier, adjuvant and delivery systems may well result in a level of protection even greater than that seen with the single mimotope.     

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti‐HCV sera

Conformational B‐cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti‐E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15‐mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2–CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti‐E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B‐cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B‐cell responses against those specific E2 epitopes that may correlate with immunity. J. Med. Virol. 82:1655–1665, 2010. 2010 Wiley‐Liss, Inc.     

Polyepitope protein incorporated the HIV-1 mimotope recognized by monoclonal antibody 2G12

A major goal in HIV-1 vaccine research is to develop an immunogen that can elicit broadly neutralizing antibodies that efficiently neutralize a wide range of the HIV-1 subtypes. Using biopanning procedure we have selected linear peptide VGAFGSFYRLSVLQS mimicking the structure of discontinuous binding sites of broadly neutralizing antibodies 2G12 from phage peptide library. As a protein carrier, we used the earlier designed artificial polyepitope immunogen named TBI (T- and B-cell immunogen), which comprises B-cell and T-helper epitopes from the HIV-1 Env and Gag proteins. On the base of selected peptide mimotope VGAFGSFYRLSVLQS the artificial protein TBI-2g12 was constructed and its immunogenic properties was investigated. It was shown that the TBI-2g12 as well as the original TBI induces antibodies that recognize HIV-1 proteins and TBI protein using ELISA and immunoblotting. However only anti-TBI-2g12 serum recognized the synthetic peptide mimotope VGAFGSFYRLSVLQS, whereas the antibodies against original TBI don't recognize it. The neutralization assay demonstrated that serum antibodies of the mice immunized with TBI-2g12 possess virus neutralizing activity. The addition of selected peptide leads to inhibition neutralizing activity of anti- TBI-2g12 serum. We conclude from these results that immunogen TBI-2g12 containing the selected peptide VGAFGSFYRLSVLQS elicits HIV-1 neutralizing antibodies during immunization. Our data suggest that this immunogen may be useful in designing effective HIV-vaccine candidates.     

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.     

A novel domain antibody rationally designed against TNF-alpha using variable region of human heavy chain antibody as scaffolds to display antagonistic peptides

Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.     

模拟HIV-1 gp41 CHR表位短肽的筛选及研究

目的 利用针对HIV-1跨膜蛋白gp41CHR序列合成肽C34的单克隆抗体1G1筛选噬菌体12肽库，旨在找寻模拟C34肽表位的序列，同时探索该短肽成为HIV-1gp41NHR与CHR结合抑制物的可能性。方法 以1G1为钓饵蛋白对噬菌体12肽库进行亲和筛选，以双夹心ELISA鉴定阳性克隆。结果 经3轮筛选后，随机挑选17个噬菌体克隆，其中6个克隆与1G1显示出较强的结合活性，上述6个阳性克隆经DNA测序，氨基酸序列相同；HYEFWAWNWEAN，其明显的疏水性质类似于G34N末端，特异性鉴定显示这些克隆均能够与HIV-1gp41N多肽结合，结论 该噬菌体克隆展示肽可模拟HIV-1gp41CHR多肽表位，并可与N多肽结合。     

Newly characterized species-specific immunogenic Chlamydophila pneumoniae peptide reactive with murine monoclonal and human serum antibodies

A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.     

A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer

Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope.     

TNF结合肽和TNFR封闭肽对TNF-α体外介导的生物学抑制效应的实验研究

目的检测TNF-α结合肽(TBP)和TNFR封闭肽(TRBP)的生物学特性及功能。方法以GFP-TNF融合蛋白竞争结合实验,检测TBP和TRBP的特异性。用非变性PAGE检测TBP和TRBP的互补结合性。用细胞毒抑制实验,观察TBP和TRBP对TNF-α杀伤U937细胞的抑制作用。用硝基蓝四氮唑(NBT)还原能力和RT-PCR技术,检测TBP和TRBP对TNF-α诱导的人外周血单核细胞的呼吸爆发和对促炎症因子IL-1β和IL-8mRNA水平的抑制作用。结果TBP和TRBP能抑制GFP-TNF融合蛋白与细胞膜上TNFR的结合。非变性PAGE证明,TBP与TRBP之间的结合,为非配体和受体的结合;TBP和TRBP对TNF-α的细胞毒效应均有抑制作用,且随着剂量的增加而增强,二者联用(即pep.38 X4)效果更好。TBP和TRBP联用,能有效地抑制TNF-α和IFN-γ诱导的单核细胞呼吸爆发的强度,抑制TNF-α诱导的IL-1β和IL-8mRNA的转录。结论TBP和TRBP具有结合TNF-α和TNFR的特异性,但二者的结合为非配体和受体的相互结合。TBP和TRBP对TNF-α介导的各种生物学效应均具有抑制作用,二者联用效果更显著。     

Cancer immunotherapy by a recombinant phage vaccine displaying EGFR mimotope: an in vivo study

Abstract

To date, several small molecule inhibitors and monoclonal-antibodies (like ICR-62) have been used to treat tumors over-expressing epidermal growth factor receptor (EGFR). However, the limitations associated with these conventional applications accentuate the necessity of alternative approaches. Mimotopes as compelling molecular tools could rationally be employed to circumvent these drawbacks. In the present study, an M13 phage displaying ICR-62 binding peptide mimotope is exploited as a vaccine candidate. It exhibited high affinity towards ICR62 and polyclonal anti-P-BSA antibodies. Following the mice immunization, phage-based mimotope vaccine induced humoral immunity. Elicited anti-EGFR mimotope antibodies were detected using ELISA method. Moreover, the phage vaccine was tested on the Lewis lung carcinoma mice model to investigate the prophylactic and therapeutic effects. The tumor volume was measured and recorded in different animal groups to evaluate the anti-tumor effects of the vaccine. Our data indicate that the reported phage-based mimotope could potentially elicit specific antibodies resulting in low titers of EGFR-specific antibodies and reduced tumor growth. However, in vivo experiments of prophylactic or therapeutic vaccination showed no specific advantage. Furthermore, phage-mimotope vaccine might be a promising approach in the field of cancer immunotherapy.     

Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.     

Transmembrane T-cell receptor peptides inhibit B- and natural killer-cell function

A synthetic hydrophobic peptide (core peptide; CP) containing two positively charged amino acids, lysine and arginine was derived from the transmembrane sequence of the T-cell receptor (TCR) alpha chain and has been shown to inhibit T-cell-mediated inflammation. In this study, we investigated the specificity of CP (10 microm) on lymphocyte function and found that it significantly inhibited interleukin-2 production in T cells and natural killer cytotoxicity by 46-58% compared to positive control. CP had no effects on B-cell proliferative responses when used at these concentrations; however, it suppressed B-cell proliferation at higher concentrations (50 microm). Inhibition by CP was not the result of membrane pore formation or cytotoxicity when examined by trypan blue, propidium iodide staining or transmission electron microscopy. CP analogues, with both lysine and arginine replaced by neutral or negatively charged amino acids, or by randomly distributing charges in the peptide sequence, had no effect on lymphocyte function. These results suggest that peptide inhibition is affected by its structure and charge interactions, and may involve common signalling molecules in T, B and natural killer cells. The potential of the immuno-inhibitory effects of CP as a novel anti-inflammatory peptide in therapy should be further explored.     

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.     

Cytotoxicity of tumor necrosis factor-alpha for human umbilical vein endothelial cells

Human umbilical vein endothelial cells were examined for sensitivity to killing by human recombinant tumor necrosis factor-alpha (TNF-alpha). Treatment of the cells with concentrations of TNF-alpha up to 50 ng/ml for 18 hours did not produce evidence of cytotoxicity. However, a marked cytotoxic effect was found when TNF-alpha pretreated cells were incubated in Hanks' balanced salt solution for a further 4 hours. Exposure of the cells to heat-inactivated or antibody-neutralized TNF-alpha did not result in cytotoxicity. Human recombinant interleukin-1 also lysed endothelial cells under the same conditions, whereas human recombinant macrophage-colony stimulating factor did not. Inclusion of superoxide dismutase, catalase, or soybean trypsin inhibitor in the culture medium during the time of endothelial cell exposure to TNF-alpha had no protective effects. Likewise, allopurinol (a xanthine oxidase inhibitor) and nordihydro-guaiaretic acid (a lipoxygenase inhibitor) were not protective under the same conditions. In contrast, the ferric iron chelator deferoxamine mesylate and three different cyclooxygenase inhibitors provided significant protection against TNF-alpha induced cytotoxicity. When human dermal fibroblasts and human squamous epithelial cells were used in place of the umbilical vein endothelial cells, these cells were resistant to TNF-alpha mediated killing. These findings demonstrate that under the experimental conditions employed, TNF-alpha is cytotoxic for human umbilical vein endothelial cells. This may have implications in a number of in vivo situations in which TNF-alpha is thought to play a role.     

Differential effects of small tumour necrosis factor-alpha peptides on tumour cell cytotoxicity, neutrophil activation and endothelial cell procoagulant activity.

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.