Golimumab, a fully human monoclonal antibody against TNFalpha

Centocor Inc and licensees Schering-Plough Corp, Mitsubishi Tanabe Pharma Corp and Janssen Pharmaceutical KK are developing golimumab, a fully human mAb antibody against TNFalpha, for the potential treatment of rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS) and ulcerative colitis. Golimumab is currently in phase III clinical trials for RA, PsA and AS and preliminary data have shown an improvement in a number of physical functions, disease activity, productivity and quality-of-life measurements.     

热休克蛋白90α、70、27mRNA在急性白血病细胞中表达的研究

运用RNA分子杂交的方法,观察了热休克蛋白(heat shock protein,HSP)90α、70、27在22例白血病病人细胞,正常血细胞及K562红白血病细胞系中的表达。结果表明:16例白血病病人中,4例急性淋巴细胞白血病(ALL),6例急性非淋巴白血病(ANLL)、1例慢性粒细胞白血病急变期(慢粒急变)和2例骨髓增生异常综合征(MDS)血细胞呈现HSP27高水平表达,较正常血细胞显著增多,ALL(5例)与ANLL(7例)白血病细胞HSP27表达水平无明显改变。检测了7例白血病细胞HSP70的表达水平,除1例ALL及1例MDS明显升高外,其余5例(包括1例ALL,3例ANLL和1例慢粒急变)显著低于正常血细胞。17例白血病病人细胞(包括9例ANLL、5例ALL、2例慢粒急变和1例MDS)HSP90α表达水平均升高,明显高于正常。结果提示:白血病细胞HSP90α表达水平的升高可能与白血病细胞活跃异常增殖有关,而HSP27基因的高表达可能不是某种急性白血病的特殊标志。     

The HSP90 Inhibitor, AT13387, Is Effective against Imatinib-Sensitive and -Resistant Gastrointestinal Stromal Tumor Models

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799-808. ?2012 AACR.     

Preclinical pharmacokinetic analysis of SNX-2112, a novel Hsp90 inhibitor, in rats

SNX-2112 is a novel Hsp90 inhibitor. The aim of this study was to investigate the pharmacokinetics, tissue distribution, plasma protein binding and excretion profiles of SNX-2112 in Sprague-Dawley rats after a single intravenous injection. The pharmacokinetic properties of SNX-2112 were examined after a single i.v. injection of 2.5, 5, and 10 mg/kg to rats, respectively. The tissue distribution and urinary, fecal, and biliary excretion patterns of SNX-2112 were investigated following a single i.v. injection of 10 mg/kg. The results suggested that the pharmacokinetic properties of SNX-2112 fitted well into a two-compartment open model with t_1/2β values of 9.96 ± 4.32, 10.43 ± 4.06, 10.41 ± 4.38 h and area under the curve values of 7.62 ± 1.03, 8.10 ± 0.77, 15.80 ± 1.00 (μg/mL) h according to the single doses of 2.5, 5, and 10 mg/kg, respectively. Approximately 0.13 ± 0.09% and 3.62 ± 0.77% of SNX-2112 were excreted via the urine and feces within 72 h, respectively; 2.59 ± 0.67% was excreted into the bile up to 24 h after a single i.v. injection of 10 mg/kg. The major elimination route was therefore through excretion in the feces. The binding rate of SNX-2112 with plasma protein was found to be concentration-dependent. In conclusion, this study first provided the full pharmacokinetic characteristics and tissue distribution of SNX-2112, which would be helpful for its clinical regiment design.     

Drug evaluation: zanolimumab, a human monoclonal antibody targeted against CD4

Merck Serono SA, under license from Genmab A/S and Medarex Inc, is developing zanolimumab, a human mAb targeted against CD4, for the potential treatment of cutaneous (CTCL) and noncutaneous (NCTCL) T-cell lymphomas. Zanolimumab is currently undergoing phase III clinical trials for CTCL and phase II clinical trials for NCTCL.     

全人源抗Met双价抗体的制备与特性分析

目的以抗HGF受体Met特异性的全人抗体Fab基因为模板，合成并表达双价抗体Diabody。方法以天然抗体库中筛选出的Fab基因为模板，分别扩增抗体的重链可变区与轻链可变区基因，经重叠拼接合成Diabody基因，克隆于原核表达载体pBAD／gⅢA中表达。在不同的培养条件下，阳性克隆经IPTG诱导表达，ELISA、SDS—PAGE、Western-blot和细胞免疫荧光分析。结果培养基成分和诱导剂的浓度对目的蛋白的表达未见有明显的影响，低温诱导能够促进可溶性蛋白的产生；但电泳与印迹分析表明，在29kD出现预期大小蛋白条带。双价抗体经表达、变性与复性和IMAC亲和纯化后，用细胞免疫荧光分析表明，Diabody能够与S114细胞表达Met的胞外区特异性结合。结论本研究制备的双价抗体能够与其抗原特异性结合，该抗体有望与其他抗体分子结合，制备双特异性抗体，为肿瘤临床诊断或治疗提供候选分子。     

Preclinical evaluation of the antimetastatic efficacy of Pentoxifylline on A375 human melanoma cell line

Melanoma is the most common malignant skin cancer, appears indestructible and is notoriously resistant to all current modalities of cancer treatment strategies. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, has shown to have radiosensitizing properties for a variety of cancers. Recently, we have shown that PTX exhibits antimetastatic and anti-angiogenic activities in B16F10 melanoma cells in vitro as well as in vivo. In the present study, we have demonstrated the anticancer and antimetastatic potential of PTX against A375 human melanoma cell line at sub-toxic doses. The results implicate that PTX at sub-toxic doses exhibited an inhibitory effect on the ability of cellular proliferation as shown by MTT and colony formation assay. It impedes migration and also induces apoptosis. A375 cells pretreated with PTX showed decrease in adhesion to both Matrigel and Collagen type IV. Further, Gelatin zymography result reveals that PTX treatment decreases the secretion of MMP2 and MMP9. Finally, PTX significantly inhibited A375 subcutaneous tumour xenograft growth without having any toxicity. Thus PTX at sub-toxic doses affected melanoma metastasis at multiple steps in vitro as well as tumour growth in vivo. These data demonstrate its antimetastatic potential and provide preclinical evidence for the development of PTX as a potential agent against metastatic melanoma.     

抗人凋亡诱导因子单克隆抗体的制备与鉴定

背景：凋亡诱导因子不仅具有氧化还原和电子传递功能,还具有促细胞凋亡功能,从而在维持细胞正常生理活动中具有重要作用。目的：制备抗人凋亡诱导因子单克隆抗体并进行生物学特性鉴定。方法：利用Ensembl数据库及DNA star软件包对凋亡诱导因子氨基酸序列进行分析,获得优势表位多肽,采用碳化二亚胺法将多肽与载体蛋白偶联制备完全抗原免疫动物,采用杂交瘤技术制备凋亡诱导因子单克隆抗体并纯化。结果与结论：间接ELISA法检测显示,免疫鼠腹水中抗人凋亡诱导因子单克隆抗体效价达到1：252400。Western blot结果显示,存在与抗原带一致的相对分子质量67000的特异条带。免疫组化检测结果显示,在结肠癌组织细胞中有棕色阳性颗粒表达,说明此抗体也可用于免疫组识化学染色。提示实验获得了高活性、高纯度、高特异性抗人凋亡诱导因子单克隆抗体。     

IgE and IgG antibody against human (recombinant DNA) insulin in patients with systemic insulin allergy

We demonstrated IgE and IgG antibodies to human (rDNA) insulin as well as to bovine and porcine insulin in the serum of two patients with systemic insulin allergy by an enzyme-linked immunosorbent assay. We also demonstrated IgE and total antibody binding to bovine insulin with the use of radioimmunoassays. Both patients had cutaneous reactivity to all three insulins. When the serum of one patient was preincubated with human, porcine, or bovine insulin, there was inhibition of binding of the patient's IgE and IgG antibodies to human insulin. The other patient had very low levels of IgE antibodies to insulin and thus only IgG inhibition was possible. Preincubation with human insulin inhibited binding of each patient's antibodies to bovine or porcine insulin. We conclude that, for these two patients, human insulin has all the antigenic determinants that bovine and porcine insulin have. Therefore, human insulin for these two patients will not eliminate insulin allergy in all patients with systemic allergy to animal insulin, because there are patients whose antibodies recognize determinants common to commercial human, bovine, and porcine insulin.     

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

Summary. Background: β₃‐Integrins are involved in platelet aggregation via α_IIbβ₃ [glycoprotein (GP)IIb–GPIIIa], and in angiogenesis via endothelial α_Vβ₃. Cross‐reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. Objectives: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on β₃‐integrins. Methods: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC₅₀ were determined by immunodot assays, enzyme‐linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb–GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. Results: We observed specific and high‐affinity binding to an intact GPIIb–GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose‐dependent fibrinogen binding to GPIIb–GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross‐reactivity with α_Vβ₃, and also demonstrated proangiogenic effects in tube formation and CAM assays. Conclusions: These Fab fragments are the first entirely human anti‐GPIIb–GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual‐specificity anti‐β₃‐integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.     

重组人白细胞介素10抗家兔同种异体皮肤移植排斥模型的建立

背景:研究表明,白细胞介素10在移植排斥反应的预防和治疗中具有一定的应用前景,有望成为一种新的临床用免疫抑制剂.动物移植模型是研究免疫抑制剂常用的实验平台,最常用的皮肤移植模型动物是小鼠,但小鼠属于小动物模型,移植技术要求较高.目的:建立家兔同种异体皮肤移植模型,观察重组人白细胞介素10抗家兔皮肤移植排斥反应的效果.方法:取家兔背部皮肤修剪成含真皮层的全厚皮片、中厚皮片和不含真皮层的薄层皮片进行家兔自体皮肤移植,随机分为固定组和非固定组,固定组用自制脖套对家兔头颈部活动进行一定限制;药物抗家兔同种异体移植排斥设重组人白细胞介素10组、环孢素A阳性对照组和生理盐水阴性对照组.采用混合淋巴细胞反应法观察家兔混合淋巴细胞反应增殖指数.在30 d内连续观察移植皮肤出现排斥的时间和移植皮片平均存活时间.结果与结论:供、受体混合淋巴细胞培养呈增殖反应,表明家兔同种异体主要组织相容性复合体不同,相互皮肤移植可发生排斥反应.移植皮片厚度以含真皮的中厚皮片为最佳;自制脖套固定可有效限制家兔脖头颈部活动,有利于移植创面的保护.实验成功建立了重组人白细胞介素10抗家兔同种异体皮肤移植排斥模型,重组人白细胞介素10具有抗移植排斥的作用.     

重组人血小板生成素治疗脓毒症相关性血小板减少症临床疗效的Meta分析

目的 系统评价重组人血小板生成素(rhTPO)治疗脓毒症相关性血小板减少症(SAT)的临床疗效. 方法 计算机检索PubMed数据库、EMbase数据库、Cochrane library数据库、中国知网数据库、万方数据库、维普数据库自建库至2019年8月15日间关于rhTPO治疗SAT的相关文献.由3名研究人员独立进行...     

Dose range evaluation of Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, in combination with amphotericin B-desoxycholate for treatment of murine systemic candidiasis

Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t(1/2)(α) of ~0.198 h and a t(1/2)(β) of ~1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis.     

Tremelimumab, a fully human monoclonal IgG2 antibody against CTLA4 for the potential treatment of cancer

Pfizer Inc is developing tremelimumab, a fully human monoclonal IgG2 antibody against cytotoxic T-lymphocyte-associated antigen, for the potential intravenous treatment of cancer. Phase III clinical trials to confirm the role of tremelimumab in the treatment of metastatic melanoma are ongoing, as are phase II trials in patients with NSCLC and colorectal cancer.     

A prospective study of the efficacy of fluconazole (UK-49,858) against deep-seated fungal infections

Fluconazole (UK-49,858) is a new bis-triazole antifungal drug that can be administered both orally and intravenously. We conducted an open clinical trial on the efficacy of fluconazole (50-100 mg) once daily in 20 non-neutropenic patients with deep-seated fungal infections. Seventeen patients (eight females, nine males, median age 56 years) could be evaluated clinically. All patients had an underlying disease, while eight also received immunosuppressive therapy. The median duration of treatment was 33 days (range 8-194 days). Clinical cure or improvement was achieved in 14 of 17 patients (82%). Nine patients were both clinically and microbiologically cured. Fluconazole was especially effective in patients with candidal infections. Two patients with cryptococcal meningitis were clinically cured; one of the two was also microbiologically cured. No serious side effects of fluconazole were encountered.     

人重组促红细胞生成素治疗脓毒症贫血的临床研究

目的 探讨人重组促红细胞生成素治疗脓毒症贫血的临床价值.方法 选择2005年6月至2006年12月天津医科大学第二医院ICU脓毒症伴有贫血患者60例,分为人重组促红细胞生成素治疗组(n=30)和对照组(n=30).所有患者均需排除肾功能不全需血液净化治疗者、消化道出血及曾经rhu-EPO治疗者.治疗组患者入院后48h起给予皮下注射人重组促红细胞生成素,每次6000U,隔日一次,连续两周;当Hb≤80 g/L,输红细胞治疗或根据病情由主诊医师决定.数据均以(-x±s)表示,计量资料用t检验,计数资料采用精确概率法检验,P＜0.05为差异具有统计学意义.结果 两组患者治疗前后血红蛋白比较,治疗前两组患者血红蛋白差异无统计学意义(P＞0.05).治疗后治疗组血红蛋白(105.87±11.48)g/L明显高于对照组(91.23±19.89)g/L(P＜0.01).治疗组输血量(2.0 ±0.47)U显著低于对照组(2.63±0.43)U(P＜0.01).两组患者死亡率差异无统计学意义(P＞0.05).结论 脓毒症合并贫血患者给予皮下注射人重组促红细胞生成素治疗可以显著提高血红蛋白水平,减少输血.     

Delineation of a cross-reactive idiotype on human autoantibodies with antibody against a synthetic peptide

Antibody against a cross-reactive idiotype (CRI) on human IgM-rheumatoid factor (RF) antibodies was induced by immunization of rabbits with a synthetic peptide ( PSL2 ) corresponding to the second complementarity-determining region (CDR), and adjacent amino acid residues of the kappa light chain of the IgM-RF Sie . The anti-peptide antibody bound efficiently to IgM-RF proteins known to share a cross-reactive idiotype, and to their isolated kappa chains. The anti-CRI was absorbed by, and eluted from, a peptide-Sepharose affinity column. The antibody activity was inhibited by the free peptide in solution. The anti-peptide antibody thus identifies a public idiotype on human IgM-RF, that is largely dependent on the primary sequence of the second CDR of the light chain. Such peptide-induced antiidiotypes of predefined specificity may facilitate studies of the molecular basis of idiotypic cross-reactions, the inheritance and somatic diversification of antibody molecules, and the regulation of the idiotype network.     

Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.