嵌合抗体技术研究进展

综述基因工程抗体中的重要组成部分———嵌合抗体的研究进展。基因工程抗体是第三代抗体,其应用日益受到重视,但是在临床治疗中人抗鼠抗体(HAMA)反应导致鼠源性抗体的应用受到了极大的限制。利用基因工程技术对鼠源性抗体进行改造,保留或增加天然抗体的特异性和主要生物学活性、减少鼠源成分,从而克服鼠源性单克隆抗体在临床应用方面的缺陷。     

以乙肝病毒核心抗原HBcAg为载体的utrophin抗体的制备

目的构建以乙肝病毒HBc为载体的带有utrophin的两个B细胞表位多肽的融合表达质粒pHBc-2UB，表达出融合蛋白并纯化，通过免疫新西兰大白兔获得抗utrophin的抗体。方法通过DNAstarⅡ模拟出utrophin的两个显著优势的B表位。用PCR法合成片段插入HBcAg序列的78位，将该合成片段克隆至pET28a载体中，构建重组表达质粒，表达出融合蛋白HBc-2UB，纯化后免疫新西兰白兔。再将该合成片段克隆至pGEX4T-2载体中，构建重组表达质粒，表达出融合蛋白GST-2UB，用于检测两个表位的抗体。结果测序结果表明重组质粒构建成功，SDS-PAGE电泳显示融合蛋白表达正确，ELISA检测到高滴度抗体。结论以HBc呈现utrophin的B表位被成功表达和纯化，获得高滴度的与两个B表位结合的抗体，为utrophin检测和DMD治疗药物研究打下了基础。     

输血前检测不规则抗体的临床意义

目的输血前检测不规则抗体的临床意义。方法66例协助配血患者,抗体由筛选细胞采用凝聚胺技术检测,抗体类型由谱细胞鉴定,交叉配血采用coombs实验。结果①抗体筛选结果阳性结果59例,其中男13例,女46例。女性30例有妊娠史,66例患者均有输血史。阴性7例。②抗体类型鉴定发现冷抗体18例,抗-D抗体29例,抗-c抗体5例,抗-E抗体4例,抗-c、抗-e抗体3例。⑤输血效果coombs实验选择相合血液,输注后效果良好。结论输血前做抗体筛查试验,检测有临床意义的不规则抗体,选择相合血液输注,避免迟发性输血相关性溶血反应发生,提高输血安全性和有效性。     

催化抗体的研究进展

催化抗体的研究进展杨日芳，恽榴红，丁振闿（军事医学科学院毒物药物研究所，北京１００８５０）免疫化学历来是集中于抗原性和抗体。抗原之间相互作用的研究。但近年来，借助于机理化学与合成化学的工作，免疫体系细胞能够产生具有新功能的抗体──催化抗体或称抗体酶（...     

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。     

单克隆抗体研究进展

随着人们对抗体结构与功能的认识不断深入,抗体不仅在抵抗外来生物物质方面有着重要作用,而且利用它本身来防治疾病更是研究热点。单克隆抗体技术的问世,使得研究和生产单抗药物成为现实。本文就单抗药物的历史、现状、存在的问题及展望做一简要综述。     

甲状腺球蛋白抗体、微粒体抗体及过氧化物酶抗体检测在诊断甲状腺疾病中的价值浅析

目的浅析甲状腺球蛋白抗体、微粒体抗体及过氧化物酶抗体检测在诊断甲状腺疾病中的作用,探求其临床价值。方法建立甲状腺疾病组和非甲状腺疾病组,运用电化学发光法和间接免疫荧光法分别对两组患者血清中TPO-Ab浓度、以及TG-Ab、TM-Ab阳性率进行检测。结果 TPO-Ab浓度、TG-Ab及TM-Ab阳性率在甲状腺疾病组和对照组之间差异具有统计学意义,P<0.01。结论三者在甲状腺疾病的诊断中具有一定的临床意义,其中后二者联合检测在由自身免疫系统引起的甲状腺疾病中更为有效。     

RIA测定TMAb和TGAb830例分析

本文分析了正常人和甲状腺疾病患者血清甲状腺微粒体抗体(TMAb)和甲状腺球蛋白抗体(TGAb) 830例测定数据。结果表明,血清TMAb和TGAb含量近似对数正态分布,正常值范围上限分别为29.59和31.39。桥本氏甲状腺炎(桥本甲炎)、原发性甲状腺机能减退(原发甲减)、原发性甲状腺机能亢进(甲亢)等自身免疫性患者中二种抗体显著升高,尤以桥本甲炎为最。亚急性甲状腺炎(亚甲炎)二种抗体亦显著增高,提示自身免疫在本病发病机理中的重要意义。     

催化性抗体

用多种策略可制备新类型的生物活性分子：催化性抗体（抗体酶）。抗体酶高专一地催化多种化学反应，可望用于性物学，化学和医药学领域。抗体酶的研究提供了生物催化的基本认识，如过渡态稳定，接近效应，一般酸碱催化，亲电和亲核反应等。     

Recent advances in the generation of bispecific antibodies for tumor immunotherapy

Bispecific antibodies are currently in clinical and preclinical development for the treatment of various cancers. Designed to direct and enhance the body's immune response to specific tumors, bispecific antibodies consist of a targeting domain (typically an antibody fragment that binds to a tumor antigen) linked to a triggering arm that is specific for a cell-surface molecule capable of mediating a phagocytic or lytic response by macrophages, natural killer cells, T-cells, or other effector cells. Recent animal studies have confirmed the therapeutic potential of bispecific antibodies; however, results from clinical trials so far have been less promising and have revealed clear limitations of these molecules, such as immunogenicity and severe side effects caused by mass release of inflammatory cytokines. A second generation of bispecific molecules, bispecific single-chain antibodies and hetero-oligomeric-engineered antibodies, has now been produced by using DNA recombinant technology; these may theoretically improve tumor targeting and minimize side effects, eventually replacing the full-length bispecific antibodies. Over the next few years, several recombinant bispecific antibodies are expected to enter clinical trials and ultimately emerge as new pharmaceutical products. This review briefly summarizes major trends in the development of bispecific antibodies for tumor therapy, and describes a rationale for the generation of novel recombinant molecules.     

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.     

Bispecific antibodies for the treatment of tumours and infectious diseases

Bispecific antibodies are in clinical and preclinical development for the treatment of various cancers and life-threatening infectious diseases. Designed to direct and enhance the body’s immune response to specific tumours and pathogens, bispecific antibodies have shown promising results in Phase I and Phase II clinical trials, leading in some cases to complete or partial responses in cancer patients. These bispecific antibodies consist of a ‘targeting’ domain, typically a fragment of a monoclonal antibody that binds to a tumour, linked to a ‘triggering’ arm that is specific for a molecule capable of mediating a phagocytic or lytic response by macrophages, natural killer cells, T-cells or other effector cells. By mediating an immune assault on tumours or pathogens, bispecific antibodies may also lead to antigen presentation and a vaccine-like response in patients. Over the next few years, we expect several bispecific antibodies to enter the late stages of clinical trials and ultimately emerge as new pharmaceutical products.     

Bispecific antibodies for the treatment of tumours and infectious diseases

Bispecific antibodies are in clinical and preclinical development for the treatment of various cancers and life-threatening infectious diseases. Designed to direct and enhance the body's immune response to specific tumours and pathogens, bispecific antibodies have shown promising results in Phase I and Phase II clinical trials, leading in some cases to complete or partial responses in cancer patients. These bispecific antibodies consist of a 'targeting' domain, typically a fragment of a monoclonal antibody that binds to a tumour, linked to a 'triggering' arm that is specific for a molecule capable of mediating a phagocytic or lytic response by macrophages, natural killer cells, T-cells or other effector cells. By mediating an immune assault on tumours or pathogens, bispecific antibodies may also lead to antigen presentation and a vaccine-like response in patients. Over the next few years, we expect several bispecific antibodies to enter the late stages of clinical trials and ultimately emerge as new pharmaceutical products.     

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.     

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.     

Therapeutic bispecific antibody formats: a patent applications review (1994-2017)

Introduction: Bispecific antibodies have become increasingly of interest by enabling new therapeutic applications such as retargeting cellular immunity towards tumor cells. About 23 bispecific antibody platforms have therefore been developed, generating about 62 molecules which are currently being evaluated for potential treatment of a variety of indications, such as cancer and inflammatory diseases, among which three molecules were approved. This class of drugs will represent a multi-million-dollar market over the coming years. Many companies have consequently invested in the development of bispecific antibody platforms, creating an important patent activity in this field.

Areas covered: The present review gives an overview of the patent literature over the period 1994–2017 of different immunoglobulin gamma-based bispecific antibody platforms and the molecules approved or in clinical trials.

Expert opinion: Bispecific antibodies are progressively accepted as potentially superior therapeutic molecules in a broad range of diseases. This frantic activity creates a maze of hundreds of patents that pose considerable legal risks for both newcomers and established companies. It can consecutively be anticipated that the number of patent conflicts will increase. Nevertheless, it can be expected that patents related to the use of a bispecific antibody will have tremendous commercial value.     

针对HER2靶点的抗体药物研究与肿瘤靶向治疗

人类表皮生长因子受体2(HER2)属于跨膜酪氨酸激酶受体家族的成员,在肿瘤细胞中存在过表达。研究显示在乳腺癌、卵巢癌、胃癌、肺癌、前列腺癌中均存在不同程度的HER2过表达。抗体靶向治疗与传统化疗相比,特异性强,毒副作用小。本文介绍了曲妥珠单抗和帕妥珠单抗的单药治疗效果和与化疗药物、激素治疗、疫苗的联合治疗效果,以及偶联药物策略,阐述了其他新型抗HER2抗体药物,特别是双特异抗体、免疫毒素以及抗体融合蛋白等研究近况,为相应的HER2抗体药物开发和临床应用提供参考。     

A method for making multispecific antibodies having heteromultimeric and common components

A method for manipulating whole antibodies to create preferentially-associating heterodimers is described. This technique relies on the simple idea of introducing bulky residues at the C_H3 dimer interface, disrupting homodimer assembly. Cavities created in opposing positions in another C_H3 domain, permits the assembly of a ‘knob-into-hole’ heterodimer, resulting in a heterodimer yield of over 95%, compared to a possible mixture of ten products by conventional approaches. Heterodimers such as these result in bispecific antibodies (BsAbs) that have wide utility in target-effector cell targeting and tumour cell-specific delivery of therapeutic agents.