Immunotoxin studies in a model of human T-cell acute lymphoblastic leukemia developed in severe combined immune-deficient mice

The transplantation of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line HSB-2 into severe combined immune-deficient (SCID) mice was found to produce a disseminated pattern of leukemia similar to that seen in humans. The iv injection of 10⁷ HSB-2 cells was associated with a universally fatal leukemia. Histopathological examination of animals revealed the spread of leukemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 monoclonal antibody (MAb) HB2 to the ribosome inactivating protein (RIP) saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immunotoxin (IT) to CD7⁺ HSB-2 target cells with an IC₅₀ of 4.5 pM. In an in vivo study, the IT was shown to significantly prolong the survival of SCID mice injected with HSB-2 cells compared to untreated control animals. This therapeutic effect was seen both with a single injection of 10 μg of IT given 7 d after the injection of HSB-2 cells, and was even more effective when IT was administered as three daily injections of 10 μg on d 7, 8, and 9. These results demonstrate the useful application of human leukemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 IT in human T-ALL.     

丝瓜籽中蛋白质生物合成抑制蛋白的分离、纯化及性质研究

丝瓜（Ｌｕｆｆａｃｙｌｉｎｄｒｉｃａ）种籽,经捣碎、抽提、硫酸铵分级沉淀,ＣＭ－５２离子交换层析,Ｓｅｐｈａｃｒｙ１Ｓ－１００分子筛,阳离子交换ＦＰＬＣ等步骤,分离到两种单链蛋白质生物合成抑制蛋白：Ｌｕｆｆｉｎ－Ａ和Ｌｕｆｆｉｎ－Ｂ。它们都是等电点接近１０的碱性蛋白,ＳＤＳ－ＰＡＧＥ测定分子量分别约为２７ｋｄ和２８ｋｄ,氨基酸组成分析表明两者具很大同源性,但免疫双扩散及ＥＬＩＳＡ检测证明两者的免疫原性有差异。Ｌｕｆｆｉｎｓ对兔网织红细胞裂解液的蛋白质生物合成有强烈的抑制作用,ＩＣ５０分别为１．４×１０－１１ｍｏｌ／Ｌ和２．０×１０－１１ｍｏｌ／Ｌ,比ＴＣＳ的２．９×１０－１０ｍｏｌ／Ｌ低得多。因而,Ｌｕｆｆｉｎｓ很有可能成为肿瘤导向药物的高效＂弹头＂。     

In vitro evaluation ofPyrularia thionin—anti-CD5 immunotoxin

The membrane-active peptide,Pyrularia thionin, purified fromPyrularia pubera, was covalently conjugated to an anti-CD5 monoclonal antibody. The membrane-active properties of thionin were not affected by the conjugation. The immunotoxin killed CD5⁺ lymphocytes in vitro at a concentration of 0.1 nmol/10⁷ cells after 2 h of incubation. The immunotoxin also inhibited the proliferation of T cells in vitro, stimulated either by mitogens or in the mixed lymphocyte reaction. It was shown by electron paramagnetic resonance of spin probes and differential scanning calorimetry that the ability of the immunotoxin to perturb the lipid phase of membranes is close to that of unconjugated thionin. The results obtained suggest thatPyrularia-thionin—anti-CD5 conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5⁺ leukemia and lymphomas.     

Anti-CD30 immunotoxins with native and recombinant dianthin 30

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein fromDianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30⁺ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus). L428 and L540 (both from Hodgkin's lymphoma). IC₅₀ values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30^– cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC₅₀ higher than nM).     

提高免疫毒素DT386-GMCSF在E.coli中表达量的研究

以人粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体（GM-CSFR）为靶向的白喉毒素（DT）与GM-CSF免疫毒素DT386-GMCSF为急性髓系白血病提供了一种新的替代治疗途径,但该蛋白在E.coli中的表达量很低,难以进行工业化生产。为探索造成其低表达的关键影响因素,对DT386-GMCSF中的GM-CSF进行了C端的截短表达,发现GM-CSF中L114编码序列可明显影响融合蛋白的表达量。在此基础上,构建了一系列突变体,发现保留1-123位氨基酸且将L114L115V116突变为G114V115T116的突变体DF123GVT的表达量高于DT386-GMCSF,且对来源于高表达GM-CSF受体的HL60细胞的肿瘤单细胞具有相似的细胞毒作用。DF123GVT突变体的获得为GM-CSFR靶向的免疫毒素的开发应用打下了基础。     

抗胃癌免疫毒素表达质粒的构建、表达及对肿瘤细胞的特异杀伤

以含单链抗体 ( Sc Fv) 3H1 1基因全长的质粒 DNA为模板 ,利用 PCR技术扩增 3H1 1 Sc Fv基因片段 ,扩增片段及绿脓杆菌外毒素 PE38表达质粒 p YR39- 1 - PE38经 H ind /N de 酶切、连接 ,转化大肠杆菌 BL2 1,构建免疫毒素的表达质粒 p YR3H1 1 - PE38.转化菌在 IPTG诱导下 ,表达免疫毒素 3H1 1 - PE38,3H1 1 - PE38经纯化、变性、复性处理后 ,通过 MTT法检测其对胃癌细胞MGC80 3的杀伤活性 .结果表明 ,3H1 1 - PE38浓度不变 ,其杀伤率在一定的范围内随作用时间的延长而增加 ,当浓度为 5× 1 0 -8mol/L,作用时间为 60 h时 ,其对胃癌 MGC80 3细胞的杀伤率达74 .2 % ,而同等条件下抗 DNA免疫毒素 p Ig2 0 - PE38的杀伤率仅为 9.2 % ;作用时间一定 ( 60 h) ,免疫毒素浓度与杀伤率呈正相关 ,在 1 0 -10 mol/L以下 ,杀伤率几乎为零 ,而浓度高于 5× 1 0 -8mol/L时 ,杀伤率超过 70 % . 3H1 1 - PE38能够有效杀伤与之特异结合的胃癌细胞 ,具有潜在的应用前景     

Expression and purification of cysteine introduced recombinant saporin

Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC₅₀ values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.     

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.