Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells

Receptor of human interleukin 4 (hlL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R,a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.     

肿瘤细胞靶向性白细胞介素4(IL-4)融合蛋白的研究

白细胞介素 4受体 (IL-4R)特异地存在于多种肿瘤细胞表面 ,这为某些肿瘤的治疗提供了一个靶向标记。在以前的研究中 ,人白细胞介素-4(Hil-4)与白喉毒素 (DT)的融合蛋白 (DT4H)被构建 ,且它对某些肿瘤细胞系的高毒性得到了证明。但是 ,由于毒素部分的强免疫原性 ,它可以诱导人体的免疫反应。该研究中我们构建了白细胞介素 4与绿脓杆菌外毒素 (PE) 253～608aa的融合蛋白 ,并在其N端添加了 6×His标记方便纯化 ,在其C端添加了KDEL提高毒性。为了改善与IL-4R的亲和力我们将IL-4进行了环式重组 ,构建的融合毒素 ,H404K ,经DEAE$CSepharoseFastFlow及Ni-NTA纯化后 ,纯度达 90 %。纯化后的H404K与DT4H相似 ,对U251高度敏感 ,对MCF7及HepG2中度敏感 ,且我们首次证实该毒性不会被兔抗白喉毒素的多克隆抗体所抑制。这些研究表明 ,H404K与DT4H可以以一种互为替代的方式用于某些恶性肿瘤的治疗     

Diphtheria toxin-related cytokine fusion proteins: elongation factor 2 as a target for the treatment of neoplastic disease

We have used protein engineering and recombinant DNA methodologies in order to construct a fusion protein in which human interleukin-2 (IL-2) is genetically linked to the catalytic and transmembrane domains of diphtheria toxin. The fusion toxin, DAB₄₈₆IL-2, is highly cytotoxic for only those cells which display the high affinity interleukin-2 receptor (IL-2R) on their surface. In phase I/II clinical studies the intravenous administration of DAB₄₈₆IL-2 has been found to be safe, well tolerated and may lead to the induction of durable remissions in patients presenting with a variety of IL-2R positive lymphomas.     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.     

白喉毒素-白细胞介素2重组嵌合毒素的克隆表达及特异性细胞毒作用的研究

应用PCR技术分别扩增出编码白喉毒素氨基端 389个氨基酸 (DT3 89)的基因片段及人IL 2全基因 ,将两基因串连插入 pET3a载体 ,构建成含有DT3 89 IL 2融合基因的表达载体 ,转化大肠杆菌BL2 1,经表达、纯化后 ,用3 H Leucine掺入法测定其对HUT 10 2细胞的蛋白合成抑制作用。SDS PAGE电泳分析表明 ,表达产物分子质量 (Mr)约为 5 8kD ;重组嵌合毒素能够特异性地抑制高表达IL 2受体的HUT 10 2细胞的蛋白生物合成 ,且有一定的剂量反应关系 ,其细胞半数抑制浓度 (IC50 )约为 3 3× 10 -11mol/L。为进一步研制特异性的抗IL 2受体高表达肿瘤和相关疾病的药物打下了基础。     

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.     

Potentiation of TNF-alpha toxicity by conjugation with ricin A-chain

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor alpha (TNF-alpha) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-alpha can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-alpha cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-alpha and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC50=4 pM) not sensitive to native TNF-alpha, and sensitive WEHI cells with substantially increased lethality (LD50=0.01 fM). This increased TNF-alpha cytotoxic activity suggests that hybrid toxins containing TNF-alpha may have therapeutic applications in the treatment of cancer.     

Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons

This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.     

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.     

中枢神经系统靶向性CuZn—SOD的构建和表达

SOD对中风等由氧自由基毒性引起的神经性紊乱有保护作用,但因血脑屏障使血液中的SOD不能进入中枢神经系统。靶向性SOD可能是进入该系统的途径之一。将人CuZn-SODcDNA与破伤风毒素C部分基因融合,分别整合进pET－22b（＋）及pFastBacHTb载体中,并分别在E.coli及粉纹夜蛾Tn-5B1-4细胞中表达。表达产物分子量为68kD,与理论计算值。蛋白质印迹实验证实,其表达产物能与人CuZn－SOD多克隆抗人本及抗破伤毒素全毒互抗体有免疫反应。在Tn细胞中高效表达,表达产物占可溶性总蛋白质的20％,表达产物有SOD活性,且具有逆行轴突运输的能力。这为靶向性SOD的进一步应用创造了条件。     

Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice

Fusion proteins composed of tumor binding agents and potent catalytic toxins show promise for intracranial therapy of brain cancer and an advantage over systemic therapy. Glioblastoma multiforme (GBM) is the most common form of brain cancer and overexpresses IL-13R. Thus, we developed an interleukin-13 receptor targeting fusion protein, DT(390)IL13, composed of human interleukin-13 and the first 389 amino acids of diphtheria toxin. To measure its ability to inhibit GBM, DT(390)IL13 was tested in vitro and found to inhibit selectively the U373 MG GBM cell line with an IC(50) around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13 antibody, but not by control antibodies. In vivo, small U373 MG glioblastoma xenografts in nude mice completely regressed in most animals after five intratumoral injections of 1 microg of DT(390)IL13 q.o.d., but not by the control fusion protein DT(390)IL-2. DT(390)IL13 was also tested against primary explant GBM cells of a patient's excised tumor and the IC(50) was similar to that measured for U373 MG. Further studies showed a therapeutic window for DT(390)IL13 of 1-30 microg/injection and histology studies and enzyme measurements showed that the maximum tolerated dose of DT(390)IL13 had little effect on kidney, liver, spleen, lung and heart in non-tumor-bearing immunocompetent mice. Together, these data suggest that DT(390)IL13 may provide an important, alternative therapy for brain cancer.     

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae , a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor–toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.     

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

Synthesis and Cytotoxic Profile of a Diphtheria Toxin-Neurotrophin-4 Chimera

Abstract: A diphtheria toxin-neurotrophin-4/5 (NT-4/5) chimera (DAB₃₈₉-NT4), in which the native receptor binding domain of diphtheria toxin was replaced with a synthetic gene encoding rat NT-4/5, was expressed, refolded, and purified. This fusion toxin has a deduced molecular mass of 60,163 and is formed by joining the first 389 amino acids of diphtheria toxin to amino acids 1–130 of mature rat NT-4/5, using an NH₂-terminal bridge of 33 additional amino acids including six consecutive histidines. Neural cell types expressing only p75^LNGFR or p75^LNGFR and full-length or truncated TrkB were used to evaluate the cytotoxic efficacy of DAB₃₈₉-NT4. The fusion toxin produced a concentration-dependent killing of all cell populations, with LC₅₀ values that largely reflected the known NT-4/5 binding affinities for these receptor proteins. Mean LC₅₀ values ranged from 2,960 p M in p75^LNGFR-expressing neuro-2a neuroblastoma cells to 1,075 and 70 p M , respectively, in hippocampal astrocytes (p75^LNGFR+/truncated TrkB⁺) and cerebellar granule cells (p75^LNGFR+/TrkB⁺). The LC₅₀ for DAB₃₈₉-NT4 in receptor-negative 3T3 fibroblasts was 20 n M . NT-4/5 and brain-derived neurotrophic factor but not ciliary neurotrophic factor added in excess neutralized DAB₃₈₉-NT4 cytotoxicity. NT-4/5, however, did not reduce the cytotoxicity of intact diphtheria toxin.     

POTENTIATION OF TNF-α TOXICITY BY CONJUGATION WITH RICIN A-CHAIN

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor α (TNF-α) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-α can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-α cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-α and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC₅₀=4 pM) not sensitive to native TNF-α, and sensitive WEHI cells with substantially increased lethality (LD₅₀=0.01 fM). This increased TNF-α cytotoxic activity suggests that hybrid toxins containing TNF-α may have therapeutic applications in the treatment of cancer.     

炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

A naturally occurring genetic variant of human XRCC2 (R188H) confers increased resistance to cisplatin-induced DNA damage

Homologous recombination, a major double strand break repair pathway, plays critical roles in maintaining genome stability. Genetic polymorphisms in HR genes have been implicated in cancer risk. We report a novel assay system for evaluating polymorphisms in human homologous recombination genes using a panel of chicken DT40 repair mutants. We established mutant cell lines complemented with either wild-type or variant cDNAs of three human genes, RAD51, XRCC2, and XRCC3, and assessed their sensitivity to cisplatin and mitomycin C. DT40 mutants complemented with RAD51 coding and 5'UTR variants, and with a XRCC3 coding variant showed equivalent sensitivity as those with wild-type cDNAs. Interestingly, Xrcc2(-/-) DT40 cells complemented with variant XRCC2 (R188H) were more tolerant to cisplatin than those with wild-type XRCC2. Considering that the XRCC2 (R188H) allele reduces risk to epithelial ovarian cancer, the increased XRCC2 activity with the R188H polymorphism may have clinical benefit in preventing cancer risk.     

Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.