纳米载药系统在肿瘤多药耐药中的应用

当今世界,肿瘤已经成为威胁人类健康的重大疾病。在肿瘤疾病中,化疗可控制肿瘤的生长和转移,增强放疗的疗效,是治疗肿瘤疾病的主要手段之一。而肿瘤多药耐药是影响化疗药物疗效、引起化疗失败的重要原因,影响肿瘤患者的治愈效果,降低生存率。如何提高化疗的疗效,延长肿瘤患者的寿命成为医学界的难题。纳米载药系统是生物医学领域研究的热点,相对于单一药物,纳米载药体现了许多优越性,具有良好的应用前景。纳米级颗粒更有利于药代动力学,这些纳米载药颗粒通过被动和主动的机制表现出在全身血液循环寿命延长,持续的药物释放动力,使其能更好的在肿瘤细胞中积累而发挥作用,提高化疗的疗效。本文综述了肿瘤多药耐药研究中主要的纳米载体以及它们在逆转多药耐药方面的应用,并展望载药系统的有更多更好的发展趋势。     

基于蛋白质组学的单克隆抗体药物质谱分析

单克隆抗体药物是一类以免疫球蛋白G的结构为基础的大分子蛋白类药物,为心血管疾病、自身免疫疾病、病毒感染及癌症等多种疾病的治疗提供了全新的途径。单抗药物的迅速发展及其在多种疾病治疗中日益凸显的重要性都对单抗药物的研发及用药安全等方面提出了更高的要求。因此,建立规范可靠的分析方法对单抗药物及其糖基化修饰进行定性、定量分析,并开展单抗药物的药代动力学及药效动力学研究具有十分重要的意义。本综述将简要介绍基于质谱技术的对单克隆抗体药物及其糖基化修饰的定性定量分析。     

纳米抗体技术及其在疾病诊断和治疗中的应用

单克隆抗体具有特异性结合抗原的能力,已被广泛应用于疾病诊断及治疗领域.但因单克隆抗体的组织渗透能力较差、体内的保留时间较长以及制备过程繁琐,从而限制了其在临床中的应用.自1993年首次报道在骆驼体内天然存在的单链抗体(HCAb)以来,由于其可变区间VHH(纳米抗体)具有体积小、溶解度高、特异性强以及可在细菌中大量表达等优点,较之传统单克隆抗体,VHH在疾病的诊断治疗及药物开发等医学领域具有更广阔的应用前景.本文综述了:纳米抗体的骨架区及互补决定区与传统抗体重链相应区间的结构比较;纳米抗体库的构建以及运用噬菌体展示技术对VHH库的筛选;纳米抗体技术在疾病诊断中的应用及其用于分子显像的优势,以及纳米抗体作为抗肿瘤免疫偶联物的靶向组分在癌症治疗领域中的最新进展.     

生物技术药物药代动力学研究的方法学和实验设计

生物技术药物由于自身具有的特点 ,使得其在体内的药代动力学机制比传统药物更为复杂。近年来 ,对于这类药物在体内的代谢机制的研究日趋增加 ,研究方法也日趋成熟。概述了生物技术药物的药代动力学研究方法以及实验设计的特点。     

经皮给药纳米载体及靶向系统治疗类风湿关节炎研究进展

类风湿关节炎(RA)是全世界难治性自身免疫疾病,其治疗药物虽不断发展,但病灶药物浓度达不到有效水平导致药物疗效不理想或存在各种毒副反应,因此,基于新技术、新方法研究开发针对RA的安全、高效新型制剂是必要的。研究表明,纳米技术的运用可提高药物生物利用度,经皮给药可改善口服和注射带来的毒副作用。对近年来基于经皮给药系统治疗RA利用的纳米载体进行综述,并阐述在RA病理特征中运用到的靶向策略,思考透皮制剂的改进方法,探讨新型纳米制剂研究现状及存在的问题,从而为制备新型透皮纳米制剂提供参考。     

呼吸道合胞病毒F蛋白抗体的研究进展

呼吸道合胞病毒(respiratory syncytial virus,RSV)感染,是造成婴幼儿、学龄前儿童、免疫缺陷患者、老年人等高危群体住院治疗及死亡的重要病因。目前,多个预防RSV感染的候选疫苗正处于研发中,尚无安全、有效的疫苗面世。对RSV感染的处理仍以治疗为主,使用帕利珠单抗(Palivizumab)是当前仅有的预防药物。在过去数年间出现的新型抗体药物,如多克隆抗体、单克隆抗体、纳米抗体等有些已进入了临床前或I、II、III期临床试验阶段。融合蛋白(fusion protein,F蛋白)在RSV感染过程中是不可或缺的,它介导病毒包膜与宿主细胞膜的融合。在感染过程中,F蛋白从亚稳态的融合前构象状态(prefusion fusion protein,pre F)转变为热力学稳定的融合后状态(postfusion fusionprotein,post F)。近年来,研究人员通过不断筛选,获得了多株针对pre F的抗体。与结合post F的抗体相比,这些抗体具有更强的RSV中和活性。一些更新的抗体药物候选品,在实验中显示出了效力强、药代动力学特征明显、半衰期长等特点,并能以其他途径给药,而且能降低其制备成本。现就抗RSV pre F的抗体研究进展作一概述。     

微针用于生物大分子及纳米药物透皮给药的尝试与挑战

生物大分子及纳米药物,比如,亚单位疫苗、DNA疫苗、以及针对真皮层的治疗药物,作为近年来新兴的治疗药物,在有些治疗领域有着透皮给药的需求。由于具有靶向性高,疗效显著等特点,生物大分子及纳米药物逐渐成为新的研究热点。微针作为一种新型的给药技术,不仅具有无痛、给药方便等优点,而且运用物理手段可大幅提高大分子甚至纳米药物的透皮吸收及皮层靶向,能够避过胃肠道消化作用以及肝脏首过效用。将微针技术与生物大分子药物相结合,能够同时发挥两者的优势,实现高靶向生物药物的无痛给药。本文简述微针透皮给药技术、以及生物大分子给药的研究进展,对微针技术用于生物大分子及纳米药物透皮给药的尝试研究做了介绍和总结,对存在的技术挑战进行了分析和展望。     

周期性静滴给药的稳态动力学研究

对线性一定模型的药物,周期静滴给药的动力学模型为一分段连续函数,本文对其动态动力学特征进行了研究,得到了稳态浓度的ｃ－ｔ方程以及一次给药和多次给药动力学参数之间的关系。为预测稳态浓度提供了依据,在此基础上对给药方案的拟定进行了讨论。     

细胞药代动力学研究进展

经典的药物代谢动力学理论是建立在血浆药物浓度测定的基础上,常难以真实有效地预测体内药物的药效。很多药物必须穿透多重生物屏障,与细胞内的靶点相结合才能发挥药效。因此药代动力学研究迫切需要从“宏观”的血浆药物浓度深入到“微观”的细胞/ 亚细胞水平。综述细胞药代动力学研究领域取得的进展,重点介绍细胞药代动力学理论的提出、技术体系的建立及其在药物研发、筛选、临床方面的应用。     

经皮给药载体载MD-CPT透明质酸纳米乳的细胞吞噬与体内药代动力学研究

本文在研究制备了包载10,11-亚甲二氧基喜树碱(MD-CPT)的透明质酸纳米乳(HANs)经皮给药系统的基础上,进一步研究了载MD-CPT透明质酸纳米乳的细胞吞噬,并进行了体内药代动力学分析.通过优化制备条件,得到了皮肤渗透性良好的缓释剂型.从CLSM观察到药物被细胞摄入并传递入细胞核,同时,载药纳米乳的细胞吞噬效率呈时间依赖性,不同细胞株HSF、HUVES、MCF-7、KF的细胞吞噬率略有不同.用Rhodanmine B标记HANs,通过荧光显微镜观察到载药纳米乳透过角质层到达真皮层的拟动态过程.利用HPLC检测MD-CPT血药浓度,测得经皮给药半衰期T1/2是静脉注射的3.6倍,肌肉注射的1.6倍,体内药物滞留时间显著增加;血药浓度峰谷值差异小,曲线平缓,说明经皮给药能保证血药浓度呈现可控的持续性.最终通过活体成像系统和组织切片荧光显微镜,直观地反映出经皮给药后药物在大鼠体内的分布情况和各组织器官药物含量,确定载药纳米乳主要采取胞间渗透的扩散方式,在局部给药的区域滞留时间较长,有利于对浅表性的病灶区持续给药,延长药效,而剩余的MD-CPT和解离的HANs都进入了血液循环,最终通过新陈代谢被排出体外.为无创型HANs经皮给药系统应用于浅表性肿瘤治疗提供了理论基础.     

A nanobody directed to a functional epitope on VEGF,as a novel strategy for cancer treatment

Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.     

纳米抗体的稳定性及其结构基础研究进展

驼类纳米抗体结构简单、易于改造,且具有低免疫原性、高稳定性、高特异性、高亲和力等特点,因而具有广泛的应用前景。纳米抗体的优势之一在于其具有较高的稳定性,比常规抗体更易于储藏和运输,甚至在高温、化学和压力等极端条件下变性后仍可有效地重折叠并恢复其抗原亲和力。本文综述了纳米抗体稳定性与其结构基础方面的研究进展,阐述了纳米抗体氨基酸序列、二硫键、结构域等与其稳定性的关系,揭示了高度稳定性的纳米抗体普遍具有的结构特征。基于这些结构特征,讨论了几种纳米抗体的稳定性优化策略,包括共有序列驱动的序列修复、替换易于修饰的氨基酸、净蛋白质电荷的改变、非天然二硫键的引入以及CDR超变区的移植。预期对纳米抗体的稳定性调控提供理论指导,以拓展其作为治疗药物、诊断试剂和生物传感器等方面的应用。     

Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.     

Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes

Vibrio cholerae is considered one of the major health threats in developing countries. Lack of efficient vaccine, short incubating time of the disease, and bacterium ability to survive in aquatic environment have made cholera one of the most epidemic diseases yet known. The lipopolysaccharide is one of the bacterium key antigens used to classify V. cholerae into 206 serogroups. V. cholerae serogroup O1 is a causative agent of all cholera pandemics. Research has shown that anti-lipopolysaccharide (LPS) antibodies could provide protective immunity in cholera cases. In this research, we used N-terminal fragments of the camel's heavy-chain antibodies called VHH or nanobodies and produced a phagemid library. The obtained library was panned against V. cholerae O1 LPS, and four monoclonal nanobodies were isolated. Isolated nanobodies were tested in LPS ELISA and bacterial ELISA. The nanobody with the highest affinity toward the bacterium was used in an in vivo challenge and successfully neutralized the bacterium infection. The isolated nanobody showed high thermostability and proteolytic resistance in characterization tests.     

Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

抗体偶联药物的技术现状和展望

抗体偶联药物（antibody drug conjugate,ADC）通常由抗体通过链接体与毒素小分子偶联而成,同时具备抗体的高靶向性和小分子药物的高活性,使之作为一种新兴的靶向治疗手段,在肿瘤治疗领域展现出了优秀的疗效和潜力,成为药物研发领域的新热点。目前全球已有14款ADC药物获批上市,处于临床研究阶段的ADC候选药物分子超过140个。为了进一步提高ADC药物的安全性和有效性,近年来涌现出了各种新颖的技术。本文对ADC药物分子的关键元素,包括抗体、链接体、毒素小分子以及偶联技术等方面的最新研究进展进行总结,并讨论其优缺点。期望这些讨论能够帮助增加对ADC药物研究和开发更加系统的理解,为研发出更加高效和安全的ADC药物带来一些思考。     

Applications of nanobodies in plant science and biotechnology

Key message

Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens.

Abstract

Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

     

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (?30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.