Modification of ricin A chain, by addition of endoplasmic reticulum (KDEL) or Golgi (YQRL) retention sequences, enhances its cytotoxicity and translocation

 A pKK expression system in Escherichia coli was used to produce recombinant ricin A chain (rRTA) and rRTA modified by addition of organelle-specific amino acid retention sequences, including KDEL (an endoplasmic reticulum, ER, lumen retention signal), KKMP (an ER membrane retention signal), YQRL (a trans-Golgi network retention signal) and KFERQ (a lysosome-targeting signal) to the C terminus of rRTA. The toxicities of these RTA mutants were assessed in Jurkat cells following fluid-phase endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytotoxic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This difference did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles have been employed to assess translocation of rRTA and mutant rRTA. RTA-KDEL and RTA-YQRL respectively exhibited 6.7-fold and 6.1-fold more protection against papain digestion in reconstituted ER vesicles and 2.2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, than unmodified rRTA. These mutants were reassociated with ricin B chain to form holotoxins. The mutant RTA-KDEL and RTA-YQRL holotoxins were 3.8-fold and 1.5-fold more cytotoxic for target cells, respectively, than ricin produced using unmodified rRTA. Our results suggest that both ER and the trans-Golgi network may play important roles in the intracellular trafficking and translocation of ricin A chain. Received: 14 August 1997 / Accepted: 14 October 1997     

人血管生成素-PE40融合蛋白基因的构建、表达及活性研究

利用 PCR扩增出人血管生成素 (h ANG)成熟肽的基因片段 .与绿脓杆菌外毒素缺失突变体PE40的基因连接后 ,克隆入 p UC1 9载体中 .测序后克隆入表达载体 p RSETB,构建成 h ANG-PE40融合基因的表达载体 .IPTG诱导 ,表达出分子量约为 58k D的 His6- ANG- PE40融合蛋白 ,占菌体总蛋白的 8% .Ni2 +- NTA树脂纯化表达蛋白 ,SDS- PAGE结果显示纯化重组蛋白为单一条带 .鸡胚绒毛尿囊膜鉴定表明重组蛋白体外能够有效地抑制血管的形成 .     

In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified fromMomordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC₅₀ = 1–10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC₅₀ = 10 pM). Moreover, the in vitro performances of the immunotoxin compared favourably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model ofnu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%,P <0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.     

The Different Behavior of Diphtheria Toxin, Modeccin and Ricin in HeLa Cells Infected with Trypanosoma cruzi

ABSTRACT. We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi . Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.     

Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence

Background

Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen (¹O₂).

Methods

The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab–saporin, was evaluated in combination with PCI using TPCS_2a (Amphinex®), a new photosensitizer approved for clinical use.

Results

PCI synergistically enhanced the cytotoxicity of trastuzumab–saporin on trastuzumab-resistant HER2⁺ Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment (“light after” strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS_2a-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by ¹O₂ generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane.

Conclusions

Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins.

General significance

PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the “light first” procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.     

白喉毒素类免疫毒素研究进展

白喉毒素类免疫毒素是将缺失天然受体结合活性的白喉毒素片段或突变体与抗体或细胞因子偶联而得到的一类新型导向药物,它可特异性识别并结合靶细胞,通过发挥其ADP核糖基化活性而抑制细胞蛋白合成,引发细胞凋亡。由于白喉毒素类免疫毒素能高效、特异地杀伤特定靶细胞,而使其在肿瘤等疾病的药物开发中暂露头角。综述了基于白喉毒素的免疫毒素的研制现状与应用前景 。     

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor β

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor β (FRβ) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FRβ-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRβ monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FRβ-expressing macrophages will provide a therapeutic tool for human glioblastomas.     

Production and introduction of a novel immunotoxin based on engineered RNase A for inducing death to Her1-positive cell lines

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein–protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose–response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC₅₀ of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use.     

T lymphocyte killing by a xanthine-oxidase-containing immunotoxin

Summary We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radicalproducing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested.     

Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster-5A antigen associated with human small-cell lung cancer

Summary The cytotoxic properties of a ricin A chain immunotoxin made with the mouse monoclonal antibody SWA20, recognising a family of sialoglycoprotein antigens selectively expressed by human small-cell lung cancer (SCLC), were examined using a panel of tumour cell lines in tissue culture. SWA20—ricin-A-chain was selectively toxic to the SW2, NCI-H69 and GLC-8 SCLC cell lines, inhibiting the incorporation of [³H]leucine by 50% at a concentration of 0.2–2 nM, but had no selective activity against the NCI-H23 and NCI-H125 lung adenocarcinoma or the control CEM T-lymphoblastoid cell lines. The SWA20 immunotoxin intoxicated the SW2 cell line rapidly, inhibiting [³H]leucine incorporation by 50% within 2 h compared with 0.5 h for ricin. Analysis of the effects of SWA20—ricin-A-chain on the growth of SW2 cells using a limiting-dilution clonogenic assay revealed that the immunotoxin could eliminate 95% of clonogenic malignant cells. Although SWA20—ricin-A-chain was found to be rapidly active against the majority of tumour cells, its action was limited by the presence of insensitive cells expressing low levels of the target antigen.