双特异性抗体研究进展

抗体药物具有特异性高、不良反应少的特点, 已成为肿瘤治疗的热点。双特异性抗体(双抗)是指同时结合2个不同抗原或同一抗原2个表位的抗体。与单特异性抗体相比, 双抗具有高度特异性, 增强了治疗有效性和安全性, 同时减少了不良反应。已有多种双抗药物获批上市, 进入临床研究的双抗药物数量不断增加。双抗药物的形式持续在更新, 应用范围也在扩大, 在未来具有非常好的应用前景。此文就目前已有的双抗类型、作用机制及目前双抗制备上面临的困难等进行概述。     

双特异性抗体研究进展

抗体药物具有特异性高、不良反应少的特点,已成为肿瘤治疗的热点。双特异性抗体(双抗)是指同时结合2个不同抗原或同一抗原2个表位的抗体。与单特异性抗体相比,双抗具有高度特异性,增强了治疗有效性和安全性,同时减少了不良反应。已有多种双抗药物获批上市,进入临床研究的双抗药物数量不断增加。双抗药物的形式持续在更新,应用范围也在扩大,在未来具有非常好的应用前景。此文就目前已有的双抗类型、作用机制及目前双抗制备上面临的困难等进行概述。     

双特异性抗体研究进展

抗体药物具有特异性高、不良反应少的特点,已成为肿瘤治疗的热点。双特异性抗体（双抗）是指同时结合2个不同抗原或同一抗原2个表位的抗体。与单特异性抗体相比,双抗具有高度特异性,增强了治疗有效性和安全性,同时减少了不良反应。已有多种双抗药物获批上市,进入临床研究的双抗药物数量不断增加。双抗药物的形式持续在更新,应用范围也在扩大,在未来具有非常好的应用前景。此文就目前已有的双抗类型、作用机制及目前双抗制备上面临的困难等进行概述。     

去唾液酸糖蛋白受体单链抗体靶向蜂毒肽的体外抑瘤效应研究

目的探讨抗去唾液酸糖蛋白受体(ASGPR)单链抗体C1与蜂毒肽(Melittin)重组蛋白C1M靶向抑制肝癌细胞的效果。方法将含C1M/pGC的XL1-Blue转化菌通过IPTG诱导表达。表达产物用Ni2+螯合柱亲和纯化,免疫组织化学技术分析重组蛋白C1M的抗原结合能力,四氮唑盐(MTT)法检测C1M对肝癌细胞株HepG2的增殖抑制作用。结果原核表达质粒C1M/pGC在XL1-Blue中获得有效表达;表达产物以可溶性形式存在;纯化的C1M相对分子量为29.4×103;免疫组化结果表明C1M能有效识别去唾液酸糖蛋白受体,与HepG2(C1M)共培养3d,细胞生长抑制率达92.0%。结论在大肠埃希菌中成功表达C1M,位于C端的Melittin蜂毒肽能有效抑制肿瘤细胞的生长,为体内研究Melittin的靶向抗肝癌效果奠定了基础。     

抗EGFR/抗KDR双特异性单链抗体的构建及表达

针对EGFR或VEGFR信号通路的相互作用导致的耐药性,为增强抗EGFR或抗VEGFR2抗体单独使用疗效不佳的癌症的治疗效果,本文利用重叠PCR将抗人EGFR胞外区单链抗体基因scFv-E10和抗人VEGFR2(KDR)的单链抗体基因scFv-AK404R融合在一起,得到抗EGFR/抗KDR双特异性单链抗体(bispecific single-chain diabody,scDb)基因,scDb的连接肽序列为15个氨基酸(G4S)3,在肽链的C端引入His标签及myc标签。将scDb基因连接入载体pHEN2,转化大肠杆菌HB2151并诱导表达,经镍柱纯化获得电泳纯scDb蛋白,产量约为570μg.L 1发酵液。ELISA测定显示,scDb与EGFR胞外区的结合与亲本scFv-E10相当,与KDR胞外区的结合略低于亲本scFv-AK404R,表明抗EGFR/抗KDR scDb可与EGFR和KDR两种抗原特异性结合,可用于进一步的抗肿瘤活性研究。     

浅谈二甲双胍的抗肿瘤作用

二甲双胍是目前治疗2型糖尿病的一线口服药.除了有效发挥降糖作用外,流行病学研究和动物实验表明,二甲双胍还具有抗卵巢癌等抗肿瘤的作用,其可以降低卵巢癌发病率、提高患者生存率.本文就近年来二甲双胍与肿瘤发展和治疗的临床研究进展和潜在机制进行了综述,将为糖尿病肿瘤患者的治疗提供新的思路.     

单链双价抗体连接肽的优化设计△

以单链抗体为元件构建的基因工程双价抗体分子近年来在肿瘤的治疗方面显示出良好的应用前景,但双价单链抗体分子的亲和性及抗肿瘤活性分别较衍生因子的功能及活性有所变化。合适的连接肽可以赋予双价单链抗体更适于临床应用需求的生物学活性,如何选取或设计连接肽是构建单链双特异性抗体的关键步骤。在分子量子化的模型基础上,运用同源模建的方法建立单链抗体分子间结构与功能的关系模型,采用改进的遗传算法把连接肽的设计看作为方程的求解问题,改善算法的搜索能力、搜索效率和收敛性能,设计全局最优的连接肽。双价单链抗体连接肽的设计是在充分利用生物信息学的基础上,探索具有不同功能域的复合蛋白质以及连接肽的设计,为恶性肿瘤的靶向多功能基因治疗提供科学依据,并为寻找有效的治疗因子提供研究方向。     

二甲双胍用于治疗胰腺癌的研究进展

目的:为将二甲双胍用于治疗胰腺癌提供参考。方法:通过查阅近十余年国内外相关研究文献,就二甲双胍对胰腺癌的治疗作用及作用机制等进行归纳和综述。结果与结论:二甲双胍能降低糖尿病患者胰腺癌的发病率,延长胰腺癌患者的生存时间,增加癌细胞对放化疗的敏感性。其抗胰腺癌的主要作用机制包括激活AMPK、抑制炎症因子、抑制特异蛋白转录因子、抑制肿瘤干细胞等。虽然二甲双胍抗胰腺癌作用的临床前研究较为乐观,但其相关临床研究结果还存在差异,还需设计更严密的临床试验以获取更充分的证据,从而为治疗胰腺癌开辟新的途径。     

单链抗体的研究进展及其在疾病治疗中的应用

单链抗体(single chain fragment variable, scFv)是通过基因工程改造的重组抗体, 保留了抗体的靶向性, 同时因为体积较小而获得了完整抗体不具有的特性, 例如渗透性好、易扩散、免疫原性弱、体内存留时间短以及容易表达等。这些特性使得scFv可以作为一种十分理想的靶向性分子应用于疾病的诊断和治疗。scFv可以与荧光蛋白融合发挥疾病诊断的作用;也可以与化学毒素相连形成免疫毒素用于癌症的治疗;scFv连接到T细胞上, 则构建成嵌合抗原受体T细胞, 是十分具有前景的癌症治疗技术。此文综述了scFv的相关制备技术, 以及一些比较有希望的临床及临床前应用。     

单链抗体的研究进展及其在疾病治疗中的应用

单链抗体(single chain fragment variable, scFv)是通过基因工程改造的重组抗体,保留了抗体的靶向性,同时因为体积较小而获得了完整抗体不具有的特性,例如渗透性好、易扩散、免疫原性弱、体内存留时间短以及容易表达等。这些特性使得scFv可以作为一种十分理想的靶向性分子应用于疾病的诊断和治疗。scFv可以与荧光蛋白融合发挥疾病诊断的作用;也可以与化学毒素相连形成免疫毒素用于癌症的治疗;scFv连接到T细胞上,则构建成嵌合抗原受体T细胞,是十分具有前景的癌症治疗技术。此文综述了scFv的相关制备技术,以及一些比较有希望的临床及临床前应用。     

Recombinant antibodies: a novel approach to cancer diagnosis and therapy

Recombinant antibodies and their fragments currently represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. These reagents dominate the cancer-targeting field, as highlighted by the recent approval of the first engineered therapeutic antibodies by the Food and Drugs Administration (FDA). Last year, important advances have been made in the design, selection and production of recombinant antibodies. The natural immune repertoire and somatic cell affinity maturation has been superseded by large antibody display libraries and rapid molecular evolution strategies. These novel libraries and selection methods have enabled the rapid isolation of high-affinity cancer targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications. In alternative strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Antibody-directed cancer pre-targeting followed by prodrug activation (ADEPT) has proved a most promising therapeutic strategy. Multi-specific antibodies have been effective for cytotoxic T-cell recruitment and antibody-fusion proteins have delivered enhanced immunotherapeutic and vaccination strategies. The new millennium is indeed an exciting time for the design, selection and formulation of a range of new antibody-based products for cancer diagnosis and therapy.     

Recombinant approaches to IgG-like bispecific antibodies

One of the major obstacles in the development of bispecific antibodies (BsAb) has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods. In contrast to the rapid and significant progress in the development of recombinant BsAb fragments (such as diabody and tandem single chain Fv), the successful design and production of full length IgG-like BsAb has been limited. Compared to smaller fragments, IgG-like BsAb have long serum halflife and are capable of supporting secondary immune functions, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. The development of IgG-like BsAb as therapeutic agents will depend heavily on our research progress in the design of recombinant BsAb constructs (or formats) and production efficiency. This review will focus on recent advances in various recombinant approaches to the engineering and production of IgG-like BsAb.     

Microencapsulation of therapeutic bispecific antibodies producing cells: immunotherapeutic organoids for cancer management

Regardless of the important therapeutic advances developed over the last years for the management of cancer, the fact is that many patients still suffer from a tremendous reduction on their quality of life due to lack of complete selectivity of conventionally administered chemotherapeutic drugs. In the search of more efficacious tumor-targeted therapies, the use of bispecific antibodies (bsAbs) capable of simultaneous binding to tumor-associated antigens and to an activating receptor, such as CD3, has emerged as a promising approach. With the intention to complementing and improving this cancer immunotherapy, human HEK-293 cells have been genetically modified ex vivo to secrete a recombinant anti-CEA (carcinoembryonic antigen)?×?anti-CD3 bsAb. After encapsulation in alginate-poly-l-lysine microcapsules, bsAb-secreting HEK-293 cells were monitorized for several weeks. This system has proved to be feasible for the maintenance of cell growth and recombinant antibody production giving proof-of-concept of its use as immunotherapeutic organoids in cancer treatment.     

Monoclonal antibodies as targeting and therapeutic agents: prospects for liver transplantation, hepatitis and hepatocellular carcinoma

1. Monoclonal antibodies (mAbs) of high specificity and stability have become key resources in the therapeutic, diagnostic and drug discovery fields to treat various immunological disorders and malignancies of different organs. 2. The latest genetic engineering technology applied in antibody design and production, such as phage display technology and genetically modified mouse, have revolutionized the clinical applicability and feasibility of the use of mAbs in humans. 3. Innovative antibody products in the forms of single-chain or super-humanized antibody therapeutics having a higher affinity for target antigens and minimal antigenicity in hosts have been introduced for experimental purposes and/or clinical trials. 4. Although there are successful examples of antibody therapeutics in the market, the use of mAbs in treating hepatitis-related disease and hepatocellular carcinoma is rare and remains to be exploited.     

Recombinant antibodies in infectious disease

Antibody-based therapies have a proven record of efficacy against many diseases. However, passive immunotherapy is presently underdeveloped in the field of infectious diseases. The emergence of new recombinant technologies for the generation of therapeutic antibodies provides significant opportunities for the development of effective antibody-based drugs. Obstacles to the broader use of antibodies as anti-infectives include a continuous emphasis in the field on the use of microbicidal chemotherapy, the fact that antibodies are usually pathogen-specific drugs with relatively small markets, expense of production, the necessity for early and accurate diagnosis prior to use and the complex logistics necessary for therapeutic use. Nevertheless, many opportunities for developing antibody-based drugs now exist in areas where the available antimicrobial therapies are inadequate. As reflected in patents published in the years 2000 – 2003, activity in this field is increasingly focused on novel antibody drug candidates and it is likely that, in the near future, several antibody reagents will be developed for clinical anti-infective use.     

Drugs targeting integrins for cancer therapy

Background: Integrins are cell adhesion receptors involved in development, angiogenesis, blood clotting, inflammation and cancer. Abnormal integrin expression is a hallmark of cancer and angiogenic endothelial cells. Integrin-targeted therapy is, therefore, considered a promising novel treatment approach in oncology. Objective: We describe the biological background making integrins an attractive therapeutic target as well as the effects of integrin-targeted therapies in preclinical and clinical settings. Methods: A literature search in integrin-targeted therapy was conducted, focusing on α_vβ₃, α_vβ₅, α₅β₁ and α₄β₁ integrin ligands as well as in vivo models and clinical trials. Results/conclusion: Blocking certain integrins can inhibit tumor growth and integrin ligands can be used to target cytotoxic agents to cancer tissue. Clinical trials using integrin inhibitors have yielded variable results and continuing studies evaluate their role as monotherapy or in combination with chemo- or radiotherapy.     

Development and prospects for bispecific antibody-based therapeutics in cancer and other applications

Background: Progress in molecular biology for the production of bispecific antibodies (BiAbs) and the expanding knowledge on receptors on malignant and normal target cells provide the basis for developing new strategies using antibody- and/or receptor-based platform technology for the treatment of cancer and other diseases. Objective: This review provides a preclinical and clinical perspective on application of bispecific antibodies for the treatment of solid tumors, hematologic malignancies and other diseases. Methods: This review focuses on BiAb-based immunotherapy, clinical trials, alternative strategies, the challenges of technology and future applications. Results/conclusion: The successful application of a particular BiAb will depend on a thorough evaluation of the expected functional application and thoughtful engineering of structure, affinity and number of binding sites based on the desired function.     

食管癌人源噬菌体单链抗体库的构建与初步筛选

目的构建全人源化食管癌噬菌体单链抗体库,从中筛选Eca-109细胞特异性单链抗体。方法取食管癌病人癌肿周围淋巴结作为B细胞的来源,提取总RNA,用RT-PCR的方法获得抗体可变区基因cDNA文库。首先分别网格筛选确定扩增VH和VL基因片段的引物对,以cDNA为模板扩增VH和VL基因片段,再以它们为模板分别扩增VH-linker与VL-linker,用SOE-PCR技术将后者组装成scFv,再引入酶切位点SfiI和NotI。将scFv基因克隆入噬菌粒载体pCANTAB-5E后电转入EcoliTG1,经抗体表达的辅助噬菌体M13KO7超感染,构建单链抗体库。PCR鉴定阳性重组菌EcoliTG1中scFv的插入率,1.5%琼脂糖凝胶电泳鉴定SfiI和NotI双酶切质粒的结果。从该抗体库中筛选特异性识别Eca109细胞的单链抗体,将阳性克隆菌转化EcoliHB2151进行可溶性表达。抗体亲和层析纯化后经SDS-PAGE、West-ern blot鉴定,通过ELISA法、免疫组化法、免疫细胞化学鉴定其与人食管癌细胞的结合的特异性。结果食管癌周围淋巴结总RNA的提取琼脂糖电泳结果中可见清晰的28S、18S条带;VH基因的大小约为450bp,VL基因为350bp,组装后的scFv基因约为850bp。用pUC18标准质粒测定转化效率达到108cfu.μg-1。scFv的阳性插入率为91.7%(22/24)。在筛选过程中,食管癌噬菌体单链抗体得到富集,收获率逐轮得到提高,第4轮为第1轮的141倍;SDS-PAGE与Western blot结果显示抗体分子量为30ku左右,在Ecoli HB2151中实现了单链抗体的可溶性表达。免疫组织化学结果提示可溶性抗体仅食管癌组织着色,而肝癌、胃癌组织不染色。免疫细胞化学结果表明此可溶性抗体可使食管癌细胞Eca109染色。细胞ELISA测定结果显示可溶性抗体具有较高的特异性,能与Eca109细胞结合,而不与胃癌BGC-823和NHEEC结合。结论从食管癌病人癌肿周围淋巴结成功地构建人源食管癌噬菌体单链抗体库,并从中筛选到食管癌特异性抗体。     

药动学/药效学模型研究抗CD3/EpCAM双特异性抗体M701在人结肠癌异种移植小鼠中的抗肿瘤作用

M701 为抗分化簇3/表面上皮细胞黏附因子(cluster of differentiation 3/epithelial cell adhesion molecule,CD3/EpCAM)的双特异性抗体,拟用于治疗癌细胞腹腔转移引起的恶性腹水.本研究应用群体模型方法,构建M701在人结肠癌异种移植小鼠中的药动学/药...     

Stimuli-responsive nanoscale drug delivery systems for cancer therapy

Abstract

Currently, with the rapid development of nanotechnology, novel drug delivery systems (DDSs) have made rapid progress, in which nanocarriers play an important role in the tumour treatment. In view of the conventional chemotherapeutic drugs with many restrictions such as nonspecific systemic toxicity, short half-life and low concentration in the tumour sites, stimuli-responsive DDSs can deliver anti-tumour drugs targeting to the specific sites of tumours. Owing to precise stimuli response, stimuli-responsive DDSs can control drug release, so as to improve the curative effects, reduce the damage of normal tissues and organs, and decrease the side effects of traditional anticancer drugs. At present, according to the physicochemical properties and structures of nanomaterials, they can be divided into three categories: (1) endogenous stimuli-responsive materials, including pH, enzyme and redox responsive materials; (2) exogenous stimuli-responsive materials, such as temperature, light, ultrasound and magnetic field responsive materials; (3) multi-stimuli responsive materials. This review mainly focuses on the researches and developments of these novel stimuli-responsive DDSs based on above-mentioned nanomaterials and their clinical applications.