抗体药物的药学监护

近年来，抗体药物在自身免疫性疾病及肿瘤治疗领域表现非凡，成为当前药物研发的热点之一。与传统小分子药物相比，抗体药物具有结构功能复杂、药动学特殊及免疫原性等特点，使其对临床合理用药提出了新的挑战。本文以抗体药物的结构和药动学特征为基础，从抗体药物的有效性、安全性、经济性、免疫原性和药物警戒五个方面，阐述如何将药学监护手段应用于抗体药物，旨在为抗体药物的临床合理使用提供参考。     

Anti-epitope antibody, a novel site-directed antibody against human acetylcholinesterase

AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induc- tion and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody. METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChEwas chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. T…     

Monoclonal antibody technology

The development, production, limitations, and uses of monoclonal antibody (MoAb) technology are presented. The first MoAbs were developed in 1975 using a process whereby the antibody-producing spleen cells of mice that had been immunized against sheep red blood cells were fused with the cells of a mouse myeloma cell line, producing hybridomas. These hybridoma cells are used to produce MoAbs, which are antibodies that will bind to only one specific target site on an antigen. Large quantities of MoAbs are grown, either in cell cultures or in the peritoneum of mice, and harvested. Although large quantities of MoAbs can be produced, these techniques are limited because of the potential for contamination by mouse viruses and the inability of the hybridomas to yield sufficient quantities of MoAbs. MoAbs are currently used in diagnostic techniques, including pregnancy tests and drug assays, as well as in tests for detecting viral and bacterial infections and cancer. MoAbs, coupled with dyes or radioactive isotopes, can be used in imaging techniques. Other possible applications of MoAbs include tissue typing, purification, therapy of cancer and autoimmune diseases, and treatment of drug toxicities. As the use of MoAbs in health care increases, pharmacists will need to have a good understanding of the functions and applications of these agents.     

Self-buffering antibody formulations

Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer." Buffer capacities of four representative IgG(2) molecules (designated mAb1 through mAb4) were measured in the pH 4-6 range. The buffer capacity results indicated that the mAbs possessed a significant amount of buffer capacity, which increased linearly with concentration. By 60-80 mg/mL, the mAb buffer capacities surpassed that of 10 mM acetate, which is commonly employed in formulations for buffering in the pH 4-6 range. Accelerated high temperature stability studies (50 degrees C over 3 weeks) conducted with a representative antibody in a self-buffered formulation (50 mg/mL mAb1 in 5.25% sorbitol, pH 5.0) and with solutions formulated using conventional buffers (50 mg/mL mAb1 in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, also at pH 5.0) indicated that mAb1 was most resistant to the formation of soluble aggregates in the self-buffered formulation. Increased soluble aggregate levels were observed in all the conventionally buffered (acetate, glutamate, and succinate) formulations, which further increased with increasing buffer strength. The long-term stability of the self-buffered liquid mAb1 formulation (60 mg/mL in 5% sorbitol, 0.01% polysorbate 20, pH 5.2) was comparable to the conventionally buffered (60 mg/mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2) formulations. No significant change in pH was observed after 12 months of storage at 37 and 4 degrees C for the self-buffered formulation. The 60 mg/mL self-buffered formulation of mAb1 was also observed to be stable to freeze-thaw cycling (five cycles, -20 degrees C --> room temperature). Self-buffered formulations may be a better alternative for the development of high-concentration antibody and protein dosage forms.     

测定抗心磷脂抗体、抗心肌线粒体抗体在诊治扩张型心肌病中的意义

目的比较抗心磷脂抗体(ACA)和抗心肌线粒体抗体(ACMA)对扩张型心肌病(DCM)诊断及治疗评估价值。方法采用ELISA法测定DCM,30例患儿ACA-lgG和ACMA-lgG,并与健康者对照,同时检测心肌酶、心电图和多普勒超声心动图。结果DCM患儿ACA-lgG和ACMA-lgG阳性率分别为53.3%和40.0%,均显著高于健康者(P<0.01)。ACA-G阳性患儿心肌酶CK-MB、心律失常发生率、心腔扩大率、左心收缩功能降低率均显著高于ACA-lgG阴性者(P<0.05);而ACMA-lgG阳性与否同上述改变无明显相关性(P>0.05)。结论ACA-lgG阳性对DCM患者诊断和病情轻重判定均有重要意义;ACMA-lgG阳性虽对DCM患儿诊断有一定价值,但对病情判断无作用。     

Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator

Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had granted a 50/50 co-development and co-promotion option to GSK for certain therapies that complete phase IIa trials successfully. The companies subsequently entered into a definite worldwide, co-development and commercialization agreement in August 2006, under which HGS will be responsible for conducting phase III trials of the product, with assistance from GSK. The companies will share equally phase III/IV development costs, sales and marketing expenses, and profits. In October 2007, Cambridge Antibody Technology was integrated into MedImmune. Both companies were previously independent subsidiaries of AstraZeneca. MedImmune is now the operationally independent biologics business unit of AstraZeneca. Human Genome Sciences intends to initiate a phase II trial of subcutaneous belimumab by mid-2008. Results from a phase II trial in 449 patients with SLE demonstrated that belimumab improved or stabilized SLE over 2.5 years. The phase II trial was a double-blind, placebo-controlled, multicentre study that evaluated the safety, optimal dosing, and preliminary efficacy of belimumab in patients with active SLE over 52 weeks initially, followed by a continuation phase for a total of 2.5 years. Belimumab has received fast-track designation for the treatment of SLE from the US FDA and has also been selected for inclusion in the agency's continuous Marketing Application Pilot 2 programme.     

液态芯片技术检测抗人类白细胞抗原抗体抗主要组织相容性Ⅰ类相关链A位点抗体在移植肾围手术期急性抗体介导性排斥反应中的应用

目的探讨液态芯片技术(Luminex)检测移植肾受者围手术期发生急性抗体介导性排斥反应者抗人类白细胞抗原(HLA)抗体、抗主要组织相容性Ⅰ类相关链A位点(MICA)抗体水平与影响。方法选取我院器官移植研究所2008年1月至2013年6月肾移植术后围手术期患者106例为研究对象,采用Luminex技术检测接受同种异体肾移植手术并明确病理诊断肾围手术期急性抗体介导性排斥反应患者血清中抗HLA抗体、抗MICA抗体,并同步检测血肌酐、尿量及移植肾超声等临床指标。结果参照Banff 2007标准组织病理学诊断移植肾超急性排斥反应2例,急性抗体介导性排斥反应15例,同时应用Luminex方法检测抗HLA抗体、抗MICA抗体水平,HLA抗体检测结果15例阳性,阳性率为88%(15/17),检出PRAⅠ类和Ⅱ类阳性率分别为47%(7/15)和53%(8/15);MICA抗体2例阳性,阳性率为12%(2/17),其余89例其他病理类型检测未见抗HLA抗体、MICA抗体阳性,移植肾穿刺病理结果显示HLA抗体、抗MICA抗体阳性患者C4d沉积均为阳性。结论抗HLA抗体、抗MICA抗体可预测围手术期急性抗体介导性排斥反应发生及治疗效果,对于及时诊断及治疗排斥反应提供了一个重要指标,可影响移植肾的长期存活。     

单克隆抗体药物的研究进展

通过淋巴细胞杂交瘤技术或基因工程技术制备单克隆抗体药物,已经成为生物制药领域的一个重要方面,由于单克隆抗体药物专一性强、疗效显著,因此成为近年来研究的热点药物之一。此文就单抗药物的分类、应用进行了综述,并对其应用前景及存在的不足作了概述。     

Specific antibody synthesis in vitro. II. Age-associated thymosin enhancement of antitetanus antibody synthesis

A decline in T cell function accounts for many of the observed age-related deficient immune responses. Specific antibody response to many antigens requires T cell cooperation, and deficient antibody response to such antigens has been demonstrated with aging. In an effort to assess the potential reconstitutive capacity of Thymosin Fraction 5, in vitro antitetanus antibody production was measured in tetanus toxoid booster-immunized young and old volunteers. 22 young and 12 old volunteers were immunized with tetanus toxoid and plasma antitetanus antibody and in vitro lymphocyte production of antitetanus antibody was measured. Plasma antitetanus antibody response was significantly greater in the young. In vitro antitetanus antibody synthesis was negligible prior to immunization and peaked in cultures established 1 week after immunization from both young and old. When Thymosin Fraction 5 was added to the cultures, however, there was a dose-related enhancement of antibody synthesis in 7 of 10 from the group of elderly volunteers, but only 3 of 12 from the younger group. Our data indicate that specific antibody response is deficient in the elderly, but can be enhanced in vitro by thymosin. A future clinical trial of thymosin as an adjuvant to active immunization for the elderly is warranted.     

瓜氨酸合成蛋白抗体、抗RA33抗体检测在类风湿关节炎诊断中的应用

目的 探讨瓜氨酸合成蛋白抗体（CPA）和抗RA33抗体检测在类风湿关节炎（RA）诊断中的意义。方法 用ELISA法分别检测94例RA和97例非RA风湿病患者血清中的CPA、抗RA33抗体和类风湿因子（RF）水平。结果 CPA、抗RA33抗体和RF对RA诊断的敏感性分别为73．4％、31．9％和72．3％;特异性分别为96．9％、75．3％和77．3％CPA与抗RA33抗体或RF联合可提高诊断的特异性;CPA和RF滴度呈正相关。结论 CPA对RA的诊断有良好的特异性和敏感性,较抗RA33抗体和RF为优,可作为RA诊断的血清学指标,与RF联合检测效果最佳。     

The art of antibody process development

Biopharmaceutical drug development is an intricate path that spans a dozen years from discovery through registration. The development of a therapeutic antibody presents substantial challenges, particularly with respect to the creation and implementation of manufacturing process technologies. Process development and large scale biotherapeutic manufacturing is an art generally only practiced within industry. As a consequence, these technologies may be seen as something of a 'black box' by many in the medical community. This article provides insight into the current art of antibody process development leading to market entry of novel, life-saving medicines.     

Evaluation of a surrogate antibody for preclinical safety testing of an anti-CD11a monoclonal antibody

Surrogate antibodies are a potential solution to the limited safety testing possible with humanized monoclonal antibodies with restricted species cross-reactivity. However, there are currently no defined criteria by which a potential surrogate antibody should be judged prior to its use in determining safety issues for the clinical agent. We propose that, potential surrogates should undergo rigorous evaluation to assess pharmacological and toxicological activities in comparison to the clinical agent. The current studies evaluated a chimeric mouse/rat anti-mouse CD11a monoclonal antibody (muM17) as a potential surrogate for efalizumab, a humanized anti-CD11a antibody in development for psoriasis. CD11a is a subunit of lymphocyte function associated antigen-1, an integrin involved in cell-cell interactions important to immune responses and inflammation. In vitro pharmacology studies included binding affinity to whole mouse blood and inhibitory activity of muM17 in a mixed lymphocyte response assay. In vivo pharmacology was examined using a delayed type hypersensitivity assay in female CD-1 mice. The toxicology evaluation included a murine tissue cross-reactivity study and in vivo multiple dose studies in female CD-1 mice which were administered muM17 (0.1-30 mg/kg) via subcutaneous injections once a week for 4 weeks. Clinical observations, body weight, clinical pathology, T cell CD11a expression, immunogenicity, toxicokinetics, and lymphoid organ histopathology were evaluated. Finally, since reproductive safety testing would be an important application of the proposed surrogate antibody, a pilot study in pregnant mice was conducted that demonstrated proportional transfer of muM17 into the fetus. These studies demonstrated that muM17 has pharmacological and toxicological activities similar to efalizumab. The selection of dose and regimen for GLP (Good Laboratory Practice) toxicology studies and extrapolation to clinical dose levels was based on pharmacodynamic activity (CD11a downmodulation on T cells).     

Glycosylation profiles of therapeutic antibody pharmaceuticals

Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry.To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market.Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.