A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes.     

Hairy cell leukemia: short review,today's recommendations and outlook

Hairy cell leukemia (HCL) is part of the low-grade non-Hodgkin lymphoma family and represents approximately 2% of all leukemias. Treatment with splenectomy and interferon-α historically belonged to the first steps of therapeutic options, achieving partial responses/remissions (PR) in most cases with a median survival between 4 and 6 years in the 1980s. The introduction of the purine analogs (PA) pentostatin and cladribine made HCL a well-treatable disease: overall complete response rates (CRR) range from 76 to 98%, with a median disease-free survival (DFS) of 16 years a normal lifespan can be reached and HCL-related deaths are rare. However, insufficient response to PA with poorer prognosis and relapse rates of 30–40% after 5–10 years of follow-up may require alternative strategies. Minimal residual disease can be detected by additional examinations of bone marrow specimens after treatment with PA. The use of immunotherapeutic monoclonal antibodies (mAB) like rituximab as a single agent or in combination with a PA or more recently clinical trials with recombinant immunotoxins (RIT) show promising results to restrict these problems. Recently, the identification of the possible disease-defining BRAF V600E mutation may allow the development of new therapeutic targets.     

抗肝癌单链抗体融合PE38重组免疫毒素的构建、表达及纯化

目的 :构建绿脓杆菌外毒素PE38融合人源化鼠抗肝癌单链抗体 (hscFv)原核表达载体 ,并对其产物作初步纯化 ,检测其对人肝癌细胞系SMMC 772 1的毒性作用 .方法 :采用基因工程原理 ,将PE38基因片段亚克隆入hscFv表达载体 pGEX 4T 1中 ,重组克隆载体经酶切及DNA序列测定证实两片段连接正确 ;转化大肠杆菌JM10 9后 ,受IPTG诱导表达GST融合蛋白 ,产物包涵体经变性、复性及重折叠并经GST亲合层析柱层析、凝血酶消化后 ,得到纯化的hscFv PE38融合蛋白 .采用MTT法检测hscFv PE38对SMMC772 1的杀伤作用 .结果 :实验成功构建了免疫毒素表达载体pGEX 4T 1 hscFv PE38(以下简称pGEXh PE38) ;诱导产物主要以包涵体形式存在 ,表达量为 11% ;纯化的hscFv PE38对SMMC 772 1细胞系具有较明显的杀伤作用 ,最大杀伤率为78% ,IC50 为 0 .386mg·L-1.结论 :人源化抗肝癌单链抗体与PE38重组免疫毒素的成功表达纯化及对人肝癌细胞系的试验结果 ,为进一步肝癌的导向治疗提供依据     

白细胞介素2-绿脓杆菌外毒素抑制小鼠角膜移植后的排斥反应

目的 观察白细胞介素2-绿脓杆菌外毒素(IL-2-PE40)对小鼠角膜移植排斥反应的抑制作用.方法 以C57BL/6小鼠为供者,Balb/c小鼠为受者,制备小鼠同种异体穿透性角膜移植模型,治疗组受者于手术当天起腹腔注射IL-2-PE40,用量为0.6μg/g,每12 h 1次,直至排斥反应发生;对照组相应时间点腹腔注射等体积生理盐水.术后每周在裂隙灯下观察移植角膜2次,根据Hori等的分级标准,对移植角膜混浊和新生血管进行分级,判断排斥反应发生情况,发生排斥反应时判定为移植角膜存活终止;并在术后第10、15、25、35天分别取术眼,行组织学观察,同时收集外周血,测定T淋巴细胞亚群及T淋巴细胞集落形成数.结果 治疗组与对照组移植角膜存活时间分别为(30.2±2.9)d和(15.1±2.1)d,对照组于术后第15天出现排斥反应;术后对照组CD4~+细胞开始升高,于第15天升高最为明显,达(63.9±4.0)%,随后下降,而治疗组CD4~+细胞仅轻微上升,第15天为(42.6±4.0)%,明显低于对照组(P<0.01);术后2个组CD8~+细胞变化不大;术后对照组T淋巴细胞集落形成数呈先升后降趋势,第15天上升最为明显,为(296±17)个,随后下降,而治疗组的变化不明显,第15天为(201±16)个,明显低于对照组(P<0.01).结论 IL-2-PE40可推迟角膜移植排斥反应的发生时间;能明显减少外周血中辅助性T淋巴细胞的作用,并能减弱小鼠外周血T淋巴细胞集落形成能力.     

Targeting the EGF receptor in breast cancer treatment

Summary Immunotoxins are a relatively new class of cytotoxic agents consisting of a catalytic toxin linked to an appropriate targeting ligand. The ligand directs the toxin to the surface of a tumor cell, whereupon the toxin enters the cell and catalytically inactivates the ribosome, thus disrupting protein synthesis and effecting cell death. Monoclonal antibodies (or their fragments) have been most commonly used to carry chemically conjugated toxins to proteins or antigens overexposed on the tumor cell surface, but specific ligands for tumor cell surface receptors could also provide effective targeting.The receptor for epidermal growth factor (EGFR) is overexpressed primarily in poor prognosis breast cancers that do not respond well to traditional therapies. Because EGFR is frequently overexpressed in breast cancer tissue and is associated with a poor prognosis, it is an attractive target for antitumor therapy.DAB ₃₈₉ EGF is an EGFR specific fusion toxin produced with recombinant DNA techniques consisting of sequences for the enzymatically active and membrane translocation domains of diphtheria toxin plus sequences for human epidermal growth factor. DAB ₃₈₉ EGF is a potent, EGFR specific, cytotoxic agent which rapidly inhibits protein synthesis by a mechanism of action similar to that of diphtheria itself. Preclinical studies in the laboratory and in animals now suggest the feasibility of investigating such an agent in the targeted therapy of patients with human breast cancer.     

Anti-B4-blocked ricin: a phase II trial of 7 day continuous infusion in patients with multiple myeloma

This phase II trial was undertaken to determine the toxicities, response rate, pharmacokinetics and frequency of human anti-mouse antibody (HAMA) and anti-ricin antibody (HARA) when the B-cell restricted immunotoxin anti-B4-bR was administered to patients with previously treated multiple myeloma (MM).
Five patients with MM were scheduled to receive a 7 d continuous infusion of anti-B4-bR. The initial four patients received therapy at 40 μg/kg lean body weight (LBW)/d. Two patients received a 7 d infusion, one patient received 6 d, and another patient 5 d of therapy. The fifth patient was treated for 7 d at a lower dose of 30 μg/kg LBW/d because of the side-effects observed in the initial patients.
Pharmacokinetic studies demonstrated a peak serum level >2.6 n M in three of the patients. Side-effects of therapy included hepatic transaminase elevations, myalgias, thrombocytopenia, nausea, vomiting, decrease in performance status, and capillary leak syndrome. One patient developed HAMA and two patients HARA. One patient developed neurologic toxicity with akinetic mutism, and died following therapy. No patient demonstrated a significant decline in M-component during therapy.
We concluded that anti-B4-bR can be administered by continuous infusion to patients with multiple myeloma, although immunotoxin levels >3 n M were associated with increased incidence of toxicity and required dose adjustment. Future trials using anti-B4-bR in MM will be needed to determine the optimal dose and administration schedule in this patient population, and to determine whether there is evidence of biologic activity.     

抗隐孢子虫ScFv-PE重组毒素原核表达载体的构建及其对虫体的杀灭作用

目的构建抗隐孢子虫ScFv-PE重组毒素原核表达载体并测定表达产物对隐孢子虫的杀灭作用。方法利用基因工程原理,以含抗隐孢子虫ScFv-PE重组毒素的菌株为模板,利用设计合成的一对引物扩增出ScFv-PE,利用pMAL-p2X载体构建重组毒素pMAL-ScFv-PE,并将其导入大肠杆菌中进行表达,产物进行SDS-PAGE和Western-blot,纯化后治疗感染隐孢子虫的小鼠。结果重组质粒经酶切鉴定正确,IPTG诱导后可看到明显的条带。凝胶薄层扫描分析目的蛋白表达量占菌体蛋白总量的21%。纯化后的表达产物治疗感染隐孢子虫的小鼠治疗组比对照组肠滞留物中卵囊计数（P〈0.05）与肠道平均累计感染积分明显减少（P〈0.01）。结论利用原核表达载体成功构建了可溶性形式表达的pMAL-ScFv-PE重组质粒,重组毒素对隐孢子虫具有明显的杀灭作用。     

Immunotoxins and other conjugates containing saporin-s6 for cancer therapy

Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface.     

甲状腺疾病的诊治现状及进展

甲状腺结节的检出率逐年增高,首要问题是明确及鉴别其良恶性质,相应的治疗方法也在不断规范、改进和创新。甲状腺乳头状癌发病率越来越高,手术的适应证掌握及规范化操作,纳米碳甲状旁腺负显影技术及喉返神经探测的应用成为保护甲状旁腺,减少喉返神经损伤的重点。此外一些针对晚期甲状腺癌的靶向治疗药物也在不断探索、研究。     

Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo

Background:

Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs).

Methods:

Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging.

Results:

The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA''-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model.

Conclusion:

Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.